CN1265049A - Polyethylenimine-based biomolecule arrays - Google Patents

Polyethylenimine-based biomolecule arrays Download PDF

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CN1265049A
CN1265049A CN 98807480 CN98807480A CN1265049A CN 1265049 A CN1265049 A CN 1265049A CN 98807480 CN98807480 CN 98807480 CN 98807480 A CN98807480 A CN 98807480A CN 1265049 A CN1265049 A CN 1265049A
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biomolecule
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J·范尼斯
J·C·塔伯恩
K·莫伊尼汉
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拉普吉恩公司
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Abstract

本发明提供一种生物分子阵列,由包含表面的固体基质构成,其中表面至少部分覆盖有一层聚(乙烯亚胺)(PEI),并且PEI层分为多个由连续第二区环绕并与其邻接的分开的第一区。 The present invention provides a biomolecule array, comprising a matrix composed of a solid surface, wherein the surface at least partially covered with a layer of poly (ethyleneimine) (PEI), PEI and a second continuous layer is divided into a plurality of regions surrounded by the adjacent thereto and the first area separated. 第一区有生物分子和PEI,而第二区有PEI并且基本上没有生物分子。 The first region has a biomolecule and PEI, while the second zone is substantially free of the biomolecule and PEI. 阵列可以通过包括以下步骤的方法制备:提供包含表面的固体基质,其中聚(乙烯亚胺)(PEI)层覆盖所述表面的至少一部分,所述涂层包括多个由连续的第二区环绕并与其邻接的分开的第一区;将生物分子上样到第一区而保持第二区基本上不含生物分子。 Array may be prepared by a method comprising the steps of: providing a solid substrate comprising a surface, wherein the poly (ethyleneimine) (the PEI) layer covers at least a portion of said surface, said coating comprising a plurality of second continuous regions surrounded by and adjoining the first region separated; the sample to the first region and the second region remains substantially free of the biomolecule on the biomolecule.

Description

基于聚乙烯亚胺的生物分子阵列 Based biomolecule array polyethyleneimine

技术领域 FIELD

本发明涉及生物分子阵列,并且更具体地涉及通过覆盖在基质上的聚乙烯亚胺层排列的具有分开的含生物分子单元的阵列。 The present invention relates to an array of biomolecules, and more particularly, to an array of biological molecules containing unit arranged on a substrate by coating polyethyleneimine having a separate layer.

发明背景在分子生物学和微生物学领域中,长期以来使用含有固定于其上的生物分子的固体支持物是常见的。 BACKGROUND OF THE INVENTION In the field of molecular biology and microbiology, long solid support containing biological molecules immobilized thereon are common. 固定提供各种优点,例如允许大量样品并且容易测定大量信号系统中所用的标记。 Fixed provide various advantages, such as allowing a large number of samples and a large number of easily measurable marker signal used in the system. 例如有很多关于将核酸多聚物与珠粒结合的文献。 For example there are many literature on nucleic acid polymer bound to the beads.

一种这样的方法是使用链霉抗生物素蛋白包被的磁珠,这种磁珠可从例如Dynal of Oslo,Norway商业购买。 One such method is the use of streptavidin-biotin-coated magnetic beads, such beads may e.g. Dynal of Oslo, Norway commercially available from. 这些珠粒与末端标记的生物素化的核苷酸接触。 These biotinylated nucleotides beads in contact with the end-labeled. 众所周知生物素与链霉抗生物素蛋白以非共价但高度稳定的方式相互作用,这对于许多目的与共价结合功能相同。 Known streptavidin-biotin and avidin interacts non-covalently but highly stable manner, which for many purposes the same binding function covalently. 因此,寡核苷酸可以通过此生物素-链霉抗生物素蛋白相互作用固定在珠粒上。 Accordingly, this oligonucleotide can biotin - streptavidin interaction avidin immobilized on the beads.

另一方法依赖于碳二亚胺化学法。 Another method relies on carbodiimide chemistry. 在此基本方法上有一些变化。 There are some variations on this basic method. 例如,正如例如Kremsky,JN等,核酸研究15:2891-2909,1987中所述的,酰肼包被的珠粒通过碳二亚胺催化的偶联与羧基修饰的寡核苷酸连接。 For example, as e.g. Kremsky, JN et al., Nucleic Acids Res. 15: 2891-2909,1987 of the hydrazide beads coated with carboxyl groups via a carbodiimide coupling catalyst modified oligonucleotides. 另一在此方法上的变化使用表面覆盖有羧基的可控制的有孔玻璃或磁性聚苯乙烯珠,羧基与氨基修饰的寡核苷酸反应,如例如Ghosh,SS等,核酸研究15:5353-5372,1987和Lund V.等,核酸研究16:10861-10880,1987中所述。 In another variation on this method using a surface covered with a carboxyl group or a controlled pore glass magnetic polystyrene beads, a carboxyl group and an amino-modified oligonucleotides reactions, such as e.g. Ghosh, SS et al., Nucleic Acids Res. 15: 5353 -5372,1987 and Lund V. et al., nucleic Acids Res. 16: 10861-10880,1987 said.

另一方法使用聚乙烯亚胺(PEI)层包裹珠粒。 Another method polyethyleneimine (PEI) beads were wrapped layer. 见例如,Wasserman,BP等,生物技术和生物工程XXII:271-287,1980;Povey,AC等,制药科学杂志75:831-837,1986;Rounds,MAJChrom.362:187-196,1986;Van Ness,J.等,核酸研究19:3345-3350,1991和PCT国际公开WO94/00600。 See, for example, Wasserman, BP, etc., Biotechnology and Bioengineering XXII: 271-287,1980; Povey, AC et al., Journal of Pharmaceutical Sciences 75: 831-837,1986; Rounds, MAJChrom.362: 187-196,1986; Van . Ness, J et al., nucleic Acids Res. 19: 3345-3350,1991 Publication WO94 / 00600 and PCT international. 而且,PEI已用于在大量各种非珠粒固定支持物和表面上固定各种分子包括核酸多聚物。 Furthermore, PEI has been used in a large variety of molecules fixed on the support of various non-fixed beads and the surface comprises a nucleic acid polymer. 见例如,Schurer,JW等,组织化学和细胞化学杂志25:384-387,1977;Alpert,AJ等,J.Chrom.185:375-392,1979;Vanecek,G.等,生物化学年鉴121:156-169,1982;El Rassi,Z.等,Chromatographia 19:9-18,1984;Rounds,MAJChrom.362:187-196,1986;Swann、WE等的欧洲专利申请No.0,197,784 A1,1986;D'Souza,SF生物技术通讯8:643-648,1986;Povey,AC等,制药科学杂志76:201-207,1987;Ngo,TT等的美国专利4,753,983,1988;Crane,LJ等的欧洲专利申请No.0,403,700 A1,1990;Trinh.CKDie Angewandte Makromol.Chemie 212:167-179,1993;Bruil,A.等,J.Biomed.Mat.Res.27:1253-1268,1993;PCT国际公开No.WO95/02184。 See e.g., Schurer, JW et al., Journal of Chemical tissue and Cytochemistry 25: 384-387,1977; Alpert, AJ, etc., J.Chrom.185:. 375-392,1979; Vanecek, G et al., Biochemistry Yearbook 121: . 156-169,1982; El Rassi, Z, etc., Chromatographia 19: 9-18,1984; Rounds, MAJChrom.362: 187-196,1986; Swann, WE, etc. European Patent application No.0,197,784 A1,1986; D 'Souza, SF biotechnology communication 8: 643-648,1986; Povey, AC et al., Journal of pharmaceutical sciences 76: 201-207,1987; Ngo, TT et al, US Patent 4,753,983,1988; Crane, LJ and other European patent applications No.0,403,700 A1,1990; Trinh.CKDie Angewandte Makromol.Chemie 212: 167-179,1993; Bruil, A, etc., J.Biomed.Mat.Res.27:. 1253-1268,1993; PCT international Publication No.WO95 / 02184. 确实,已报道PEI在无固定支持物的情况下结合寡核苷酸。 Indeed, it has been reported that PEI-bound oligonucleotides in the absence of a fixed support case.

最近,大量注意力集中在制备平坦固定支持物上的生物分子,特别是多核苷酸的阵列。 Recently, a number of biological molecules focused on the preparation of the planar fixed support, particularly a polynucleotide array. 只是作为例证的以下公开文献(以及本文引用的参考文献)提供这些生物分子阵列以及制备这些排列的方法的总体和具体综述:Eggers,MD等,DNA测序技术进展卷SPIE 1891:113-126,1993;Chetverin,AB等,生物/技术12:1093-1099,1994;Southern,EM核酸研究22:1368-1373,1994;Lipshutz,RJ等,生物技术19:442-447,1995;Schena,M.BioEssays 18:427-431,1996;Blanchard,AP等,生物传感器及生物电学11:687-690,1996;O'Donnell-Maloney,MJ等,遗传分析:生物分子工程13:151-157,1996;Regalado,A.Start-Up24-30,1996年10月和Stipp,D.财富30-41页,1997年3月31日。 Just as these exemplary arrays of biomolecules following publications (and references cited herein) as well as methods for preparing these general arrangement and specific Summary: Eggers, MD, etc., DNA sequencing technology progress volume SPIE 1891: 113-126,1993 ; Chetverin, AB et al., bio / technology 12: 1093-1099,1994; Southern, EM nucleic Acids Res. 22: 1368-1373,1994; Lipshutz, RJ et al., biotechnology 19: 442-447,1995; Schena, M.BioEssays 18: 427-431,1996; Blanchard, AP, etc., biosensors and bio-electricity 11: 687-690,1996; O'Donnell-Maloney, MJ et al., genetic analysis: biomolecular Engineering 13: 151-157,1996; Regalado , A.Start-Up24-30, October 1996 and Stipp, D. wealth pages 30-41, March 31, 1997.

已发展的关于将生物分子固定到珠粒和其它固定支持物上的专门技术中,很多报道的制备这些阵列的方法已有描述。 Specialized techniques for immobilizing biomolecules on beads and other fixed supports have been developed in many reported methods for preparing such arrays have been described. 有些令人惊讶地,至今还没有使用PEI以阵列模式固定生物分子的报道。 Somewhat surprisingly, has not yet been fixed using PEI reported biomolecule array mode. 最多,作为理论上的可能性,已提出PEI可用于使寡核苷酸与尼龙支持物连接。 Up, the theoretical possibility has been proposed PEI oligonucleotides may be used to support the nylon. 见例如,PCT国际公开No.WO 95/09248,引用Van Ness,J.等,核酸研究19:3345-3350,1991。 See, for example, PCT International Publication No.WO 95/09248, cited Van Ness, J et al., Nucleic Acids Res 19: 3345-3350,1991.

如此处公开的,本发明人已认识到与制备基于PEI的寡核苷酸阵列相关的问题,并且已解决了这些问题,因此目前基于PEI的阵列已不再是理论上的可能性。 As disclosed herein, the present inventors have recognized that the problem based on oligonucleotide arrays associated with PEI prepared, and have solved these problems, there is currently no longer PEI-based arrays are theoretical possibility. 而且,与也在此处公开的其它类型阵列相比,本发明的基于PEI的阵列提供各种优点。 Moreover, compared with other types of arrays are also disclosed herein, the present invention provides various advantages of PEI-based arrays.

发明概述本发明提供一种生物分子阵列,包括:包含表面的固体基质;所述表面至少部分覆盖有一层(聚乙烯亚胺)(PEI);所述涂层包括多个由第二区环绕并与其邻接的分开的第一区;所述第一区有生物分子和PEI;并且所述第二区有PEI并且基本上没有生物分子。 Summary of the Invention The present invention provides a biomolecule array, comprising: a solid substrate comprising a surface; the surface at least partially covered with a layer (polyethylene imine) (the PEI); said coating comprises a plurality of second regions, and surrounded by separate first region adjacent thereto; said first region has a biomolecule and PEI; and the second zone is substantially free of the biomolecule and PEI.

如此处所用的,第一区也称为点样区,因为在某种意义上生物分子上样到第一区内的PEI上。 As used herein, also referred to as a first region spotted area, because PEI loaded onto the first region on the sense biomolecules. 也如此处所用的,第二区也称为间隔区或邻接的间隔区,因为实际上间隔区作用为点样区反应生物分子之间的非反应性间隔。 Also used herein, also referred to as a second spacer region or a region adjacent to the spacer, the spacer is actually acting as a non-reactive spacer reaction between biomolecule spotted area.

在另一方面,本发明提供一种制备生物分子阵列的方法。 In another aspect, the present invention provides a method of preparing arrays of biomolecules. 此方法包括:提供包含表面的固体基质,其中聚(乙烯亚胺)(PEI)层覆盖所述表面的至少一部分,所述涂层包括多个由连续的第二区环绕并与其邻接的分开的第一区;将生物分子上样到所述的第一区而保持所述的第二区基本上不含生物分子。 This method comprises: providing a solid substrate comprising a surface, wherein the poly (ethyleneimine) (the PEI) layer covering at least a portion of said surface, said coating comprising a plurality of second continuous regions surrounded by adjacent thereto and separated a first region; biomolecule loaded onto a second region to the first region is maintained substantially free of the biomolecule.

参照附图和下面详细的描述,本发明的其它方面将变得显而易见。 And the following detailed description with reference to the accompanying drawings, the other aspects of the invention will become apparent.

附图描述图1A是根据本发明实施方案所述的阵列的俯视平面示意图。 DESCRIPTION OF THE DRAWINGS FIG. 1A is a top plan view of the embodiment of the array according to the present invention.

图1B是图1A的阵列的剖面示意图。 1B is a schematic cross-sectional view of the array of FIG. 1A.

图2A是用于制备本发明阵列的移液装置的等轴视图。 FIG 2A is an isometric view of an array of the pipetting device of the present invention was prepared.

图2B是根据本发明所述的移液头的实施方案的放大正面立视图。 2B is an enlarged front elevational view of an embodiment of the pipette tip of the present invention.

图3是带有锥形设计的另一种移液头的正面立视图。 FIG 3 is a pipette tip with another front elevational view of a tapered design.

图4A是根据本发明另一实施方案所述的带有凹槽、锥形设计的移液头的另一实施方案的正面立视图。 4A is another embodiment of the present invention with an elevational view, a front recess of another embodiment of a pipette tip of the tapered design of the embodiment.

图4B是图4A的移液头的仰视平面图。 4B is a bottom plan view of the pipette tip of Figure 4A.

图5表示根据本发明制备并用Vector Blue(Vector Laboratories,Burlingame,California)显色且用CCD相机和显微镜拍摄的微点阵列。 Figure 5 shows the preparation of the present invention is developed with Vector Blue (Vector Laboratories, Burlingame, California) and a micro-dot array taken with a CCD camera and microscope.

图6表示同时存在于单一阵列单元中的两种不同寡核苷酸如何根据本发明得到鉴定和部分定量测定。 Figure 6 shows how two different exist in a single array unit oligonucleotides nucleotide been identified and partially quantified according to the present invention.

图7表示用本发明方法学由机器人制备的阵列的CCD相机图像,其中点区域直径约100-150微米,相邻点与点之间中心到中心的间距为200微米。 7 shows a CCD camera image of an array produced by a robot to learn the method of the present invention, wherein the spot area diameter of about 100-150 microns, and the center point between the adjacent dots to-center spacing of 200 micrometers. 直径的标准偏差是约15%。 Diameter standard deviation of about 15%.

图8是用荧光滤镜在荧光下摄制的显微照片,显示从分析溶液转移的非媒介物成分(在此情况下是荧光微球体)的可重复点样(通过肉眼观察测定)。 FIG 8 is filmed with a fluorescent filter at fluorescence micrograph showing repeatable spotting (measured by visual inspection) from the analysis of the non-vehicle component of the solution was transferred (in this case a fluorescent microspheres) of.

发明详述本发明提供一种生物分子阵列。 DETAILED DESCRIPTION The present invention provides a biomolecule array. 阵列包括包含表面(优选地是平的)的固体基质,其中表面至少部分覆盖有一层聚(乙烯亚胺)(PEI)。 The array comprising a surface comprising a (preferably flat) a solid substrate, wherein the surface at least partially covered with a layer of poly (ethyleneimine) (PEI). PEI层包括多个由连续第二区环绕并与其邻接的分开的第一区。 PEI layer comprises a plurality of the second continuous region surrounds the first region and separate adjacent thereto. 第一区有生物分子和PEI,而第二区有PEI并且基本上没有生物分子,即每2000μm2第二区少于约103个生物分子。 The first region has a biomolecule and PEI, while the second zone is substantially free of the biomolecule and PEI, i.e. each second region 2000μm2 biomolecule less than about 103.

图1A是根据本发明实施方案所述的生物分子阵列10的俯视平面示意图,而图1B是其剖面示意图。 FIG 1A is a top plan schematic view of arrays of biomolecules according to embodiments of the present invention 10, and FIG. 1B is a schematic cross-sectional view. 阵列10具有带平坦表面22的固体基底或支持物基质20,并且阵列10有覆盖支持物基质20的至少一部分的表层30。 Array substrate 10 having a solid support matrix or planar surface 22 of the belt 20, and the array 10 has a cover 30 support matrix at least a portion of the surface 20. 表层30优选地由PEI构成,并且底层20优选地由坚固材料制成,PEI与底层20共价结合。 Surface 30 is preferably composed of PEI, and the bottom layer 20 is preferably made of a strong material, PEI 20 and the underlying covalently bound. PEI层30有多个点样区32(例如图1A中所示的12个)位于邻接间隔区34之中。 PEI layer 30 regions 32 with a plurality of sample points (e.g., 12 shown in FIG. 1A) being located adjacent to the spacer 34. 每个点样区32是分开的分离的区域,其特征是生物分子物质40点样在PEI层上,而间隔区的特征是PEI层30的剩余区域。 Each sampling area 32 is separated separate regions, wherein the biomolecule substance 40 spotted on PEI layer, and wherein the remaining region of the spacer layer 30 PEI. 例如,每个点样区30可以具有从表层30的表面向表层30内伸入中等深度“d”的截短的圆锥形状。 For example, each of the sampling area 30 may have the shape of a truncated cone extends into the middle of the depth "d" from the surface of the surface layer 30 to the inner skin 30. 如下面详细描述的,生物分子40扩散到PEI层30构造中并与PEI层30内的反应性胺位点共价结合。 As described in detail below, biological molecules 40 diffuse into the layer of PEI 30 is configured and combined with the reactive amine sites within covalent PEI layer 30.

PEI层30、点样区32和点样区32之间的间隔的规格一般根据特定应用变化。 32 specification interval between the PEI layer 30, the sampling area 32 and the sampling area generally vary depending upon the particular application. PEI层30的厚度“T”例如可以小到PEI分子的单层。 PEI layer 30 of thickness "T" may be, for example, as small as a single layer of PEI molecules. 另外,PEI层30表面的点样区一般直径约10-100μm,而点样区32之间的中心到中心距离“D”一般在100μm-1,000um之间。 Further, PEI layer 30 typically spotted surface area diameter of approximately 10-100μm, and the center point 32 between the center of the sample area to the distance "D" is generally between 100μm-1,000um. 在优选实施方案中,点样区32的表面直径约50μm,并且点样区32由约200μM的中心到中心距离D间隔开。 In a preferred embodiment, the surface diameter of about 32 spotted area 50 m, and the sampling area 32 from the center-to-center spacing of about 200μM apart by a distance D. 固体基质基质优选地是玻璃或硅。 Solid matrix substrate is preferably a glass or silicon. 然而,基质选择性地可以是石英、金、合成的有机聚合物如聚乙烯、聚酯薄膜和6,6-尼龙或它们的复合材料。 However, the substrate optionally may be quartz, gold, synthetic organic polymers such as polyethylene, polyester film and nylon 6,6 or a composite material thereof. 在优选实施方案,基质由分布在平坦表面的加样孔或其它凹槽构成。 Preferred embodiment, the substrate constituted by a flat surface distribution of loading holes or other recesses. 基质可以具有非渗透材料的表面形态或者它可以是渗透的如尼龙。 The substrate may have a surface morphology of impermeable material or it may be permeable such as nylon. 基质可以是膜或胶片(例如聚酯薄膜或金)。 The substrate may be a film or film (e.g. a polyester film or gold). 基本上可以使用任何允许被含有PEI但不含生物分子的区域包围的含PEI和生物分子的区域形成和保留的固体基质。 Basically any area but is allowed to be contained PEI and PEI-containing biomolecule biomolecules free region surrounded by a solid matrix formation and retention.

用于此目的的固体支持物实例是一般用于电子工业中构建半导体的硅片。 For this purpose the solid support is a general example constructed in the electronics industry for a semiconductor wafer. 这些硅片在一侧是高度磨光和反光的并且可容易地使用硅烷化学法用聚乙烯亚胺涂敷。 The silicon wafer was highly polished and reflective side and can be easily used with polyethyleneimine silane chemical coating method. 这些硅片在商业上可从诸如WaferNet(San Jose,CA)的公司得到。 These wafers are available from companies such as WaferNet (San Jose, CA) commercially. 玻片也可以用反光性涂层涂敷。 Slides may be coated with a reflective coating. 具有反光性涂层的玻片也可容易地使用硅烷化学法用聚乙烯亚胺涂敷。 Slides having reflective coating may also be easily coated with polyethyleneimine using silane chemistry.

聚乙烯亚胺多聚物涂层允许使用商业上可得到的交联剂(Pierce,Rockford,IL)将寡核苷酸、PCR片段或扩增子、DNA分子或片段或其它含有胺基的生物分子共价结合到固体支持物上。 Polyethyleneimine polymer coating allows the use of commercially available crosslinking agents (Pierce, Rockford, IL) oligonucleotides, PCR fragments or amplicon, DNA molecule, or a fragment containing amine groups or other biological molecule is covalently attached to the solid support. 聚乙烯亚胺(PEI)涂敷的载片也具有长保存期稳定性的额外好处。 Polyethyleneimine (PEI) coated carrier sheet also has the added benefit of long shelf life stability. PEI涂层的制备用于将PEI层附着到基质上的化学方法大部分取决于基质的化学特征。 PEI prepared for the coating layer is attached to the PEI chemistry on the matrix consists largely depends on the chemical characteristics of the substrate. 现有技术提供大量可以将PEI附着到固定支持物上的适当化学方法实例。 The prior art provides a large number of PEI can be chemically attached to the appropriate instance on the fixed support. 例如当基质是6,6-尼龙时,PEI涂层可以用Van Ness,J等,核酸研究19:3345-3350,1991和PCT国际公开WO 94/00600中公开的方法涂敷,这两篇参考文献在此引用作为参考。 When, for example, when the substrate is nylon 6,6, PEI coating may be used Van Ness, J et al., Nucleic Acids Res. 19: 3345-3350,1991 coating method, and PCT International Publication WO 94/00600 disclosed in both of these references hereby incorporated by reference. 当固体支持物是玻璃或硅时,涂敷PEI的适当方法见例如Wasserman,BP生物化学和生物工程XXII:271-287,1980和D'Souia,SF生物技术通讯8:643-648,1986。 When the solid support is glass or silicon, the PEI coating suitable method, see e.g. Wasserman, BP Biochemistry and Bioengineering XXII: 271-287,1980 and D'Souia, SF BIOTECHNOLOGY 8: 643-648,1986.

优选地,PEI涂层与固定支持物共价结合。 Preferably, PEI coating bound covalently to the fixed support. 当固定支持物是玻璃或硅时,PEI涂层可以使用硅烷化学法与基质共价结合。 When the fixing support is a glass or silicon when, PEI coating may be a silane chemically bound to the substrate covalently. 例如,具有反应性甲硅烷氧基末端基团的PEI可从Gelest,Inc(Tullytown,PA)商业购买。 For example, PEI having reactive siloxy terminal groups may be, Inc (Tullytown, PA) is commercially available from Gelest. 可以将这样的反应性PEI与玻片或硅片接触,轻微搅拌后,PEI将附着在基质上。 Can be contacted with such reactive PEI slide or silicon wafer, after gentle stirring, PEI will adhere to the substrate. 选择性地,可以使用双功能甲硅烷基化试剂。 Alternatively, you can use a bifunctional silylating reagent. 根据此方法,玻璃或硅基质用双功能甲硅烷基化试剂处理以提供具反应性表面的基质。 According to this method, glass or silicon substrate is treated with the bifunctional silylating reagent to provide the substrate with a reactive surface. 然后将PEI与反应性表面接触并通过双功能试剂与表面共价结合。 PEI is then contacted with the reactive surface, and covalently bound to the surface through the bifunctional reagent.

PEI涂层已广泛用于结合生物分子的领域。 PEI coatings have been widely used in the field of biological binding molecules. 由于各种原因,PEI在此情况下非常有效。 For various reasons, PEI is very effective in this case. 例如,PEI是高度亲水的并且浸润含有生物分子的水溶液。 For example, PEI is a highly hydrophilic and infiltration of an aqueous solution containing a biomolecule. 另外,PEI含有许多氨基,这些氨基可以与生物分子中的酸性基团形成盐。 In addition, PEI contains many amino group, an amino group which may form salts with biomolecules acidic group. 然而,从PEI接受生物分子水溶液的容易程度可以确切地看到为什么至今它还未用于制备生物分子阵列。 However, the ease of acceptance from an aqueous solution of PEI biomolecule can see exactly how far it has not been used to prepare arrays of biomolecules. 当将生物分子水溶液点样到PEI层上时,溶液迅速吸入PEI涂层而不是留在分开的区域中。 When spotted onto PEI layer biomolecules aqueous solution rapid inhalation PEI coating rather than remain in a separate area. 因为溶液吸收,以阵列模式上样的溶液完全汇合在一起并形成单一反应性区域,这显然是在生物分子阵列制备中要避免的情况。 Because the absorbent solution to the sample on the array pattern was completely merge together and form a single reaction region, this is clearly the case in the manufacture of an array of biomolecule to be avoided. 布列溶液本发明人也公开了如何用PEI涂层制备生物分子阵列。 Cloth column was present invention also discloses how an array of biomolecule coatings prepared with PEI. 本发明方法使用一种也称为“布列溶液”的组合物,包含基于组合物总体积约35体积%到约80体积%浓度的增稠剂、生物分子(优选地是0.001μg/mL到10μg/mL浓度范围的寡核苷酸)和水。 The present invention uses a method referred to as "cloth out solution" composition comprises% by volume based on the total volume of the composition in a concentration of from about 35 to about 80% by volume thickening agent, biological molecule (preferably 0.001μg / mL to 10μg / mL concentration range oligonucleotides) and water. 令人吃惊地发现在水性寡核苷酸组合物中含有增稠剂时,增稠剂赋予组合物所需的流变学特性,因此使组合物能与此处公开的改进的弹性探头一起使用,在仅一次从组合物池汲取组合物的情况下将多个均匀的微滴移至有PEI涂层的平坦表面上。 Surprisingly been found that the thickener is contained in the aqueous composition of the oligonucleotide, the thickening agent to impart the desired rheological properties of the composition, so that the composition can be used together with the herein disclosed improved elasticity probe in the case where only one dip composition from the composition of the pool to a plurality of uniform droplets move on a flat surface with a coating of PEI.

对于液体增稠剂如甘油,浓度为35%V/V到80%V/V。 For liquid thickeners such as glycerol, at a concentration of 35% V / V to 80% V / V. 组合物中优选的增稠剂浓度在一定程度上取决于进行排列时的温度。 The preferred composition depends on the temperature at which the thickener concentration are arranged to some extent. 排列温度越低,需要使用的增稠剂浓度越低。 The lower the temperature is arranged, the lower the concentration of thickening agent to be used. 温度和粘度控制的组合使得可以在大多数固体支持物(例如玻璃、硅片、尼龙6/6、尼龙膜等)上进行排列。 The combination of temperature and viscosity control may be arranged such that the majority of the solid support (e.g. glass, silicon wafers, nylon 6/6, nylon film, etc.) on.

增稠剂的存在还有其它好处,即允许各种其它低浓度物质与生物分子同时存在。 There are other benefits of the presence of thickening agents, which allows a variety of other substances in low concentrations and biological molecules exist. 例如,0.001%V/V到1%V/V的去污剂可存在于布列溶液中。 For example, 0.001% V / V to detergent 1% V / V may be present in the cloth out of solution. 这是有用的,因为PCR缓冲液含有少量的Tween-20或NP-40,并且直接从PCR排列核酸样品而无须事先纯化扩增子通常是有利的。 This is useful because PCR buffer contains a small amount of Tween-20 or NP-40, and are arranged directly from the nucleic acid sample without prior purification of PCR amplicons is generally advantageous. 使用增稠剂允许在布列溶液中含有盐(例如NaCl,KCl或MgCl2)、缓冲液(例如Tris)和/或螯合剂(例如EDTA)。 Allow thickener containing a salt (e.g. NaCl, KCl, or MgCl2) in the cloth out solution, a buffer (e.g. Tris), and / or chelating agents (e.g. EDTA). 使用增稠剂还有其它好处,即允许交联剂和/或有机溶剂存在于布列溶液中。 Thickener has the additional advantage of allowing a crosslinking agent and / or an organic solvent solution is present in the cloth out. 正如商业上得到的,交联剂通常溶解于有机溶剂如DMSO、DMF、NMP、甲醇、乙醇等溶剂中。 As commercially obtained, cross-linking agent is typically dissolved in a solvent an organic solvent such as DMSO, DMF, NMP, methanol, ethanol and the like. 当使用增稠剂时,常用的有机溶剂可以0.05%到20%(V/V)的水平用于本发明的布列溶液中。 When using a thickener, an organic solvent may be used level of 0.05% to 20% (V / V) solution for the cloth out the present invention.

一般来说,增稠剂赋予布列溶液增加的粘度。 Typically, thickeners imparting cloth column was increased viscosity. 当布列溶液中达到适当粘度时,点样的第一滴基本上与例如第100滴大小相同。 When the solution reached the appropriate column cloth viscosity, spotting droplets substantially the same as the first example droplet size of 100. 当布列溶液中使用不适当粘度时,点样的头几滴显著大于后来点样的液滴。 When the fabric used in the solution column inappropriate viscosity, spotted drops significantly greater than the head later spotted droplets. 所需的粘度在纯水与纯甘油的粘度之间。 Desired viscosity between the viscosity of pure water and pure glycerin.

本发明的布列溶液可用于将微滴点样到几乎任何表面上。 Bridgetown solution of the present invention may be used droplets spotted onto virtually any surface. 因为固体支持物的表面特性对微滴的点样几乎没有影响或没有影响,所以生物样品可排列到几乎任何类型的有涂层表面或有聚合物涂层的固体支持物上。 Since the surface properties of the solid support has little effect on the sample droplet dot or no effect, so that the biological sample may be arranged almost on the coating surface with a solid support or any type of polymeric coating. 例如,一般的水溶液在用亲水性聚合物如聚(赖氨酸)或聚(乙烯亚胺)涂敷的固体支持物上倾向于迅速扩散而这些相同溶液不容易点样在疏水性表面如硅片上。 For example, an aqueous solution generally tend to rapidly spread the same solution which is not easy on hydrophilic polymers such as poly (lysine) or poly (ethyleneimine) coated solid support, as spotted hydrophobic surface the wafer. 然而,含有根据本发明所述的增稠剂的布列溶液可用于将均匀的微滴点样于任何这些基质上。 However, according to the thickener-containing solution of cloth out of the present invention can be used in a uniform droplets spotted on any of these substrates.

在排列过程中包括增稠剂如甘油的另一重要好处是质量控制。 In the arrangement comprises a further important advantage of the process of thickening agents such as glycerin quality control. 例如当将甘油用于本文所述的排列方法时,将一小滴液体点样于固体支持物上。 For example when glycerol is used for arranging method described herein, a small drop of the liquid spotted on the solid support. 以此处所述的方法中常用的浓度时,此甘油浓度足以防止微滴的蒸发。 In the methods described herein when used in a concentration, the glycerol concentration sufficient to prevent evaporation of the droplets. 因此,在化学处理阵列之前可检查每个排列针头的每个印迹。 Thus, prior to the chemical treatment of each array may be arranged to check each blot needle. 观察微滴的能力实质上增强了对排列过程进行质量控制的能力。 Ability to observe droplets are arranged substantially enhances the ability of quality control process. 这导致排列方法学价值的重要增加。 This arrangement results in significant increase in the value of learning methods.

生物分子可以是核酸多聚物或其类似物如PNA、硫代磷酸酯和膦酸甲酯。 Biological molecules can be nucleic acids or analogs thereof such as polymers PNA, phosphorothioates and methyl phosphonates. 核酸指核糖核酸和脱氧核糖核酸。 It refers to nucleic acids RNA and DNA. 生物分子可包括非天然和/或合成的碱基。 Biomolecules may comprise unnatural and / or synthetic bases. 生物分子可以是单链或双链核酸多聚物。 Biomolecule may be single or double stranded nucleic acid polymer.

优选的生物分子是包括寡核苷酸(多达约100个核苷酸碱基)和多核苷酸(超过约100个碱基)的核酸多聚物。 Preferred biological molecules include oligonucleotides (up to about 100 nucleotide bases) and polynucleotides (over about 100 bases) of a nucleic acid polymer. 优选的核酸多聚物由15到50个核苷酸碱基形成。 Preferred nucleic acid polymer is formed from 15 to 50 nucleotide bases. 另一优选的核酸多聚物有50到1,000个核苷酸碱基。 Another preferred nucleic acid polymer has 50 to 1,000 nucleotide bases. 核酸多聚物可以是PCR产物、PCR引物或核酸双链,在此列出几个实施例。 Nucleic acid polymer may be a PCR product, PCR primer, or nucleic acid duplex, several embodiments set forth herein. 然而,如下文所述,当核酸含有伯胺时,基本上所有核酸类型可与PEI涂敷的表面共价结合。 However, as described below, when the nucleic acid contains a primary amine, substantially all of the type of nucleic acid may be covalently bound to the PEI coated surface. 布列溶液中核酸多聚物的一般浓度为0.001-10μg/mL、优选地0.01-1μg/mL、更优选地0.05-0.5μg/mL。 Typical levels of distribution in the nucleic acid polymer solution, the column was 0.001-10μg / mL, preferably 0.01-1μg / mL, more preferably 0.05-0.5μg / mL.

优选的核酸多聚物为“胺修饰的”,因为它们经修饰在核酸多聚物的5'末端含有伯胺基,优选地在核酸多聚物的伯胺与核酸部分之间有一个或多个亚甲基。 A preferred nucleic acid polymer is "amine-modified" in that they are at the 5 'end of the nucleic acid polymer containing primary amino groups modified, preferably between the nucleic acid polymer with a primary amine or a part of a nucleic acid methylene groups. 6是亚甲基的优选数目。 6 is the number of methylene preferred. 胺修饰的核酸多聚物是优选的,因为它们可通过5'-胺基与固体支持物共价连接。 Amine-modified nucleic acid polymers are preferred because they can be connected via the 5'-amino group to the solid support covalently. 可使用5'-己胺修饰的PCR引物排列PCR产物。 Using 5'-hexylamine modified PCR primers PCR products are arranged. 可在使用胺烯丙基-dUTP(Sigma,St.Louis,MO)通过切口移位导入胺之后排列核酸双链。 After the nucleic acid duplex may be arranged amine allyl -dUTP (Sigma, St.Louis, MO) amine introduced by nick translation. 用氨基烯丙基-dUTP通过聚合酶如末端转移酶或用连接酶通过将含胺的短核酸多聚物连接到核酸上可将胺导入核酸中。 -dUTP by the polymerase, such as terminal transferase with amino allyl or short amine-containing nucleic acid polymers may be attached to the nucleic acid introduced into the amine by a nucleic acid ligase.

优选地,在与PEI涂层接触之前活化核酸多聚物。 Preferably, the nucleic acid polymer activated prior to contact with PEI coating. 通过使胺功能化的核酸多聚物与多功能胺-反应性化学物如三氯三嗪(Trichlorotriazine)混合可方便地实现这一点。 By amine-functionalized nucleic acid polymer with a multifunctional amine - reactive chemical such as trichlorotriazine (Trichlorotriazine) can be easily mixed to achieve this. 当核酸多聚物含有5'-胺基时,5'-胺可与也称为氰尿酰氯的三氯三嗪反应(Van Ness等,核酸研究.19(2):3345-3350,1991)。 When the nucleic acid polymer contains a 5'-amine, also known as the 5'-amine of cyanuric chloride is reacted with trichlorotriazine (Van Ness et al., Nucleic Acids Res .19 (2): 3345-3350,1991) can be . 优选地,将过量的氰尿酰氯加入核酸多聚物溶液中,其中超过布列溶液中核酸多聚物胺数10-到100-倍摩尔数过量的氰尿酰氯是优选的。 Preferably, an excess of cyanuric chloride was added to the nucleic acid polymer solution, wherein the solution of the nucleic acid polymer amine number of 10- to 100-fold molar excess of cyanuric chloride over the columns of the cloth are preferred. 在此方法中,大多数以胺终止的核酸多聚物与一分子三氯三嗪反应,因此核酸多聚物变成以二氯三嗪终止。 In this method, the majority of amine-terminated polymer is reacted with a nucleic acid molecule of trichlorotriazine, so the nucleic acid polymer becomes terminated triazine dichloride.

本发明的有利特征是含有生物分子的布列溶液即使含有大量的三氯三嗪也可点样到PEI涂层上。 Advantageous feature of the invention is a solution containing biomolecules cloth out even if containing a large amount of trichloro triazine can be spotted onto PEI coating. 这提供了优于在进行排列之前需要从布列溶液去除偶联剂的方法的显著优势。 This provides a significant advantage of the method needs to be removed from the cloth out of a coupling agent solution prior to performing the alignment is superior.

当核酸多聚物是双链时,本发明优选的实施方案提供两条链或其中一条链含有末端氨基。 When the nucleic acid polymer is double stranded, the present embodiment provides a preferred embodiment of the invention wherein a two-chain or chains containing terminal amino group. 双链核酸多聚物可通过一个末端氨基结合到PEI涂层上,由此固定此双链多聚物。 Double stranded nucleic acid polymer may be bound through a terminal amino group to the PEI coating, thereby fixing this double-stranded polymer. 然而,因为两条链中仅有一条链与PEI涂层共价结合,所以另一链可在变性和洗涤条件下去除。 However, since only one strand of the two strands and the PEI coating is covalently bound, so the other strand can be removed under denaturing and washing conditions. 此方法提供根据本发明实施单链核酸多聚物的排列的一种方便方法。 This method provides a convenient method of embodiment according to the arrangement of the single-stranded nucleic acid polymers of the present invention. 双链核酸多聚物可例如作为PCR反应产物得到。 Double stranded nucleic acid polymer may be, for example, as a PCR reaction product.

优选地,用常用缓冲液如磷酸钠、硼酸钠、碳酸钠或Tris HCl缓冲布列溶液。 Preferably, using conventional buffers such as sodium phosphate, sodium borate, sodium carbonate, or Tris HCl buffer solution cloth out. 布列溶液的优选pH范围是7到9,其中优选的缓冲液是新鲜制备的pH8.3到pH8.5的硼酸钠溶液。 The preferred pH range of the solution is cloth column 7 to 9, wherein the buffer is preferably pH8.3 to pH8.5 freshly prepared solution of sodium borate.

为了制备一般的布列溶液,将己胺修饰的核酸多聚物以0.1μg/ml置于0.2M硼酸钠pH8.3中至50μl总体积。 For the preparation of cloth out of the general solution, hexylamine-modified nucleic acid polymer at 0.1μg / ml was placed in 0.2M sodium borate pH8.3 to a total volume of 50μl. 然后加入10μl 15mg/mL的氰尿酰氯溶液,并且让反应于25℃持续搅拌下进行1小时。 Was then added 10μl 15mg / mL solution of cyanuric chloride, and the reaction continued with stirring at 25 deg.] C for 1 hour. 加入甘油(GibcoBrl,Grand Island,NY)至56%终浓度。 Adding glycerol (GibcoBrl, Grand Island, NY) to a final concentration of 56%. 印迹技术生物分子溶液可以通过大量目前用于微细加工的技术的任意一种上样到PEI涂层上。 Blotting techniques biomolecule solution may be one kind of the sample by any of a number of techniques currently used in microfabrication onto the PEI coating. 例如,溶液可以装入喷黑印迹头中并从这样的头部喷到涂层上。 For example, the solution may be charged and smoky blots from such a head onto the coating spray head.

图2A是表示用于选择性地将分离的、可控量生物分子溶液转移到阵列10的PEI层30上的优选装置和方法的等轴视图。 FIG 2A is a diagram for selectively separated, controlled amount of biomolecule solution is transferred to an isometric view of a preferred apparatus and method of the PEI layer 30 of the array 10. 在一种实施方案中,装置带有与致动器60可操作连接的弹性探头50和与弹性探头50相反端连接的移液头70。 In one embodiment, the actuator means having an elastic probe 60 is operably connected to the probe 50 and the elastic connection 50 of the opposite ends of pipetting head 70. 弹性探头50一般包括包住偏置部件54的套壳52和活塞56,活塞有与偏置部件54相邻接的第一端57和从套壳52中伸出的第二端58。 Flexible probe 50 generally includes a sleeve 52 encasing the biasing member 54 and the piston 56, the piston 54 has a biasing member adjacent the first end 57 and second end 58 projecting from the casing 52. 套壳52可以是管状筒而偏置部件54可以是将活塞56的第二端58从套壳52推出的压缩弹簧。 Casing 52 may be tubular cylindrical and the biasing member 54 may be a second end 58 of the piston 56 is pushed out from the casing 52 a compression spring. 活塞56的第一端57因此带有与从套壳52向内放射状伸出的止动物59咬合的凸肩57a从而限制活塞56相对于套壳52的最大延伸量。 A first end of the piston 56 with the stop 57 thus extending from the animals 52 radially inwardly of the sleeve 59 engaging the shoulder 56 to limit the maximum extension 57a ​​with respect to the amount of the casing 52 of the piston. 适当的弹性探头50可从Evertt Charles(Pomona,California),Interconnect Devices Inc.(Kansas City,Kansas)、TestConnections,Inc.,(Upland,California)以及其它制造商得到。 Probe 50 may be an appropriate elasticity., (Upland, California), and obtained from other manufacturers Evertt Charles (Pomona, California), Interconnect Devices Inc. (Kansas City, Kansas), TestConnections, Inc.

致动器60优选地沿着与阵列10垂直的轴线(箭头V所示)并在与PEI层30表面平行的平面(箭头P所示)移动弹性探头50。 The actuator 60 is preferably an elastic probe 50 and moves in a plane (arrow P) parallel to the surface of the PEI layer 30 along an axis perpendicular to the array 10 (indicated by the arrow V). 因此,致动器60控制弹性探头50从而将移液头70浸入含有生物分子流体90的容器80中,将弹性探头50定位到PEI层30的所需位点上,并且将移液头70压至PEI层30的所需位点上。 Thus, the actuator 60 controls the probe 50 so that the elastic pipette tip 70 is immersed in the fluid 90 containing biomolecules container 80, to a desired site of the PEI layer 30, the pipetting head 70 and the pressure elastic probe 50 is positioned to the desired site on the PEI layer 30. 在另一实施方案中,致动器60可能仅仅与至阵列10垂直地移动弹性探头50,而另一致动器(未显示)移动阵列10和容器80以将移液头70定位至容器80或PEI层30的所需位点。 In another embodiment, the actuator 60 may move vertically with only the elastic probe 50 to the array 10, and another actuator (not shown) moves the container 80 to array 10 and the pipette tip 80 or 70 is positioned to the container desired site PEI layer 30. 致动器60优选地是自动将生物分子溶液移至PEI层30上的机器人或其它计算机控制的操作装置。 The actuator 60 is preferably automatically moved to the biomolecule solution 30 PEI layer on the robot, or other computer-controlled operating means. 此外,可将多个弹性探头50与一个致动器连接以同时将多个生物分子物质移至PEI层30上。 Further, a plurality of elastic probe 50 and the actuator can be connected simultaneously to a plurality of biological molecules on the PEI layer 30 to move.

移液头70优选地吸取足够量的生物分子流体90至其表面上以将多个的生物分子物质移至PEI层30上并形成相应的多个点样区32(图1A中所示)。 Pipetting head 70 preferably absorb a sufficient amount of fluid 90 to the biomolecule on the surface to which a plurality of biomolecule substance moves PEI layer 30 and form a corresponding plurality of sampling area 32 (shown in FIG. 1A). 图2B是根据本发明一个实施方案所述的移液头70的放大正面立视图。 2B is the enlarged front pipetting head 70 in elevational view in accordance with one embodiment of the present invention. 移液头70优选地具有截短的圆锥形状,带有端面72和多个凹槽或沟道74。 Pipetting head 70 preferably has a truncated conical shape, with the end face 72 and a plurality of grooves or channels 74. 端面72可以是从假想的交点73凹进的平面,凹进的距离“R”约在0.00001英寸和0.010英寸之间。 End surface 72 may be an imaginary point of intersection of the plane of the recess 73, the recess distance "R" between about 0.00001 inches and 0.010 inches. 并且更优选地约在0.001英寸和0.005英寸之间。 And more preferably between about 0.001 inches and 0.005 inches. 此外,凹槽74具有以约15°到120°、并且更优选地以约60°到90°的角α向端面72聚集的翼或脊76。 Further, the recess 74 having from about 15 ° to 120 °, and more preferably at an angle α of approximately 60 ° to 90 ° to the end face 72 of the wing aggregated or ridges 76.

弹性探头50、致动器60和移液头70一起运行从而在每次致动器60将移液头70压至PEI层30上时,将可控量的生物分子流体移至PEI层30上。 The elastic probe 50, the actuator 60 and the pipette tip 70 run together so that each actuator 60 when the pipette head 70 pressed onto the PEI layer 30, a controlled amount of fluid to move the biomolecules PEI layer 30 . 致动器60开始将移液头70浸入生物分子流体90的容器80中以通过毛细作用汲取和容纳较大体积的生物分子流体92(图2B)到移液头70上。 Start actuator 60 pipette tip 70 is immersed in the fluid 90 in the container 80 of biological molecule to learn and to accommodate the larger volume of biomolecules by capillary action of the fluid 92 (FIG. 2B) to the pipetting head 70. 然后致动器60将弹性探头50定位至PEI层30上。 Then the elastic actuator 60 to the probe 50 is positioned on the PEI layer 30. 在容器80中取出移液头70后,一部分移液头70上的生物分子流体92在移液头的端面72形成悬挂物94。 In the pipette tip container 80 taken out after 70, 70 biomolecule fluid on the portion of the pipette tip 92 of the pipetting head 94 end surface 72 is formed hanging objects. 然后致动器将移液头70压至PEI层上以从移液头70上的生物分子流体部分形成阵列10的单个分开离的点样区32(图1A和1B中所示)。 Then the actuator of the pipetting head pressed onto the PEI layer 70 to form a single array of dot-like regions 10 separate from the fluid fraction from a biological molecule pipette 70 on head 32 (shown in FIGS. 1A and 1B). 致动器60优选地将移液头70压至PEI层30上以使移液头70以指定量的压力接触PEI层30。 The actuator 60 is preferably pressed the pipette tip 70 onto the PEI layer 30 to make the pipette head 70 to the specified amount of pressure contact with PEI layer 30. 然而,用相同压力将移液头70连续压至PEI层30上是困难的,因为致动器60不能总是以相同高度定位移液头70并且PEI层70表面可能不一样平。 However, using the same pipette tip 70 pressure continuously pressed onto the PEI layer 30 is difficult, because the actuator 60 is not always the pipetting head 70 is positioned at the same height and the surface of the PEI layer 70 may not be the same level. 偏置部件54因此储存把移液头70压到PEI层30上产生的能量,从而允许弹性探头50在每次移液时以基本上恒定的压力接触PEI层30而不论致动器60的行程或PEI层30表面状态的微小不规则性。 The biasing member 54 thus stored pipette head 70 to the energy generated in the pressure PEI layer 30, thereby allowing the probe 50 to a substantially constant elastic pressure contact with PEI layer 30 at each pipetting whether actuator travel 60 or minute irregularities PEI layer 30 of the surface state.

因此上述移液系统提供在每次移液头70与PEI层30接触时可移送恒定点样体积的生物分子流体的装置。 Thus each device in the pipetting head 70 in contact with the PEI layer 30 may be spotted biomolecule transfer constant volume of fluid to provide the pipetting system. 应当理解应将精确、恒定体积的生物分子流体移至每个点样区的PEI层30上以在PEI层30中维持间隔区34。 It should be appreciated that the precise, constant volume of biomolecular fluid move to the PEI layer 30 to each of the sampling area in the PEI layer 30 to maintain the spacing zone 34. 点样至点样区32上的PEI层30中的生物分子流体的量一般凭经验确定,并且它是移液头70与PEI层30接触的时间、生物分子流体90的粘度、移液头70的构造以及移液头70与PEI层30之间的压力的函数。 32 spotted on the amount of PEI layer 30 to the fluid biomolecules spotted areas are generally determined empirically, and it is time PEI layer 70 and the contact 30, the viscosity of the fluid biomolecule pipetting head 90, the pipetting head 70 the structure and function of PEI 70 between layer 30 and the pipetting head pressure. 因为偏置部件54在移液头70与PEI层30之间提供了基本上恒定的压力,所以影响移至PEI层30的生物分子流体量的首要因素是移液头70与PEI层30之间接触的时间。 Because the biasing member 54 between the pipette tip 70 and the PEI layer 30 provides a substantially constant pressure, the primary factors affect the amount of fluid biomolecule PEI layer 30 is moved between the pipette tip 70 and the PEI layer 30 contact time.

图3是根据本发明所述的移液头170的另一个实施方案的正面立视图。 FIG 3 is a front elevation view of the pipette according to another embodiment of the present invention, the head 170. 在此实施方案中,移液头170具有无凹槽或翼片的截短的锥形状。 In this embodiment, the pipetting head 170 having a truncated cone shape without grooves or fins. 因此,移液头170在其锥形部分的表面容纳生物分子流体。 Thus, the pipetting head receiving biomolecule 170 on a surface thereof a fluid of the tapered portion. 虽然移液头170可用于将生物分子流体移到PEI层30上,但是一般地使用带凹槽的移液头是更理想的,因为这样的移液头容纳更多的生物分子流体。 While the pipetting head 170 may be used to move the fluid biomolecule PEI layer 30, but in general the use of fluted pipette tip is more desirable, because more of the pipetting head receiving the fluid biomolecule.

图4A是带有多个凹槽274和翼片276的移液头270的另一实施方案的正面立视图,图4B是其仰视平面图。 FIG. 4A is a front pipetting head of another embodiment with a plurality of recesses 270 and 274 of the flap 276 elevation view, FIG 4B is a bottom plan view thereof. 移液头270基本上以与上述移液头70相同的方式操作,并且因此也提供基本上相同的优点。 Pipette tip 270 are substantially the same manner as described above with the pipetting head 70 operates, and thus also provide substantially the same advantages.

上述的移液头70、170和270代表一些可用于将生物分子流体点样到PEI层30上的移液头实例。 Above 70,170 and the pipette tip 270 may be used to represent some fluid biomolecules spotted on a pipetting head instance PEI layer 30. 应当理解可对这些移液头进行多种改进,包括使用不同形状的端面设计。 It should be understood that various modifications may be made to these pipetting head comprising end surfaces of different shapes. 例如,根据具体应用,这些移液头可以具有角锥形、圆柱形、立方形或其它适当形状。 For example, depending on the application, the pipetting head may have a pyramidal, cylindrical, cubic or other suitable shape. 此外,这些凹槽可以具有除了本发明附图中所示的构造之外的其它构造。 Further, the grooves may have other configurations in addition to the configuration shown in the drawings in the present invention. 因此,移液头不一定局限于图2B-4B中说明的那些种类。 Thus, the pipetting head is not necessarily limited to those types described in 2B-4B of FIG. 排列条件和排列后的处理如上所述的布列溶液可直接用于排列过程。 Bridgetown solution conditions and arrangement of the alignment process as described above can be used directly alignment process. 也就是说,在排列核酸多聚物的优选实施方案中,在印迹步骤前没有从未反应的氰尿酰氯中纯化活化的核酸多聚物。 That is, in a preferred embodiment are arranged in a nucleic acid polymer, in no unreacted cyanuric chloride prior to the step of imprinting purified activated nucleic acid polymers. 令人惊奇地发现在排列方法的印迹步骤前不必去除过量的交联剂。 It has surprisingly been found necessary to remove the excess crosslinker prior to the imprinting step method arrangement. 也就是说,在反应混合物中过量的氰尿酰氯不影响或竞争核酸多聚物与有PEI涂层的固体支持物的共价结合。 That is, in the reaction mixture in an excess of cyanuric chloride does not affect or compete with the nucleic acid polymer is covalently bound solid support PEI coatings. 这是因为固体支持物上的胺多于将以任何给定体积(纳升到皮升)排列的氰尿酰氯分子的数目。 This is because the amine on the solid support will be greater than any given volume (nanoliter to picoliter) the number of cyanuric chloride molecules arranged.

一般让活化核酸结合到固体支持物上的反应在20到50℃进行1到20小时。 Usually activation of a nucleic acid bound to make the reaction on the solid support was subjected to 1 to 20 hours at 20 to 50 ℃. 优选地,反应时间是于25℃1小时。 Preferably, the reaction time at 25 ℃ 1 hour.

本发明的阵列在进行杂交分析中特别有用。 Array of the invention is particularly useful hybridization assay is performed. 然而,为了进行这种分析,在进行杂交步骤前必须对固体支持物上的胺加帽。 However, in order to perform this analysis, the hybridization step must be performed before the capping amine on solid support pair. 这可通过使固体支持物与0.1-2.0M琥珀酸酐反应来实现。 This may be accomplished by reacting the solid support with 0.1-2.0M succinic anhydride reactant. 优选的反应条件是70%m-pyrol和0.1M硼酸钠中的1.0M琥珀酸酐。 Preferred reaction conditions are 1.0M succinic anhydride 70% m-pyrol and 0.1M sodium borate. 一般使反应进行15分钟到4小时,其中优选的反应时间是于25℃30分钟。 The reaction is generally carried out for 15 minutes to 4 hours, wherein the preferred reaction time at 25 ℃ 30 minutes. 残余的琥珀酸酐用3X水洗加以去除。 Succinic anhydride residue to be removed by washing with water 3X.

然后将固体支持物与在pH 7-9的0.1-10.0M硼酸钠中含有0.1-5M甘油的溶液一起温育。 Then the solid support with a solution containing 0.1-5M 0.1-10.0M glycerol in sodium borate pH 7-9 were incubated together. 该步骤通过转化为单氯二嗪对任何可以共价结合到PEI表面上的二氯三嗪“加帽”。 The binding step by conversion to mono-chloro-diazine be any covalently bonded to the PEI dichlorotriazine surface "caps." 优选的条件是在pH8.3的0.1M硼酸钠中的0.2M甘油。 Preferred conditions are 0.2M sodium borate pH8.3 0.1M glycerol in. 然后可以用含有去污剂的溶液洗固体支持物以去除未结合的物质,例如微量NMP。 May then be washed with a solution containing the detergent solid support to remove unbound material, for example, trace NMP. 优选地,将固体支持物在0.01MNaCl、0.05M EDTA和0.1M Tris(pH8.0)中加热至95℃5分钟。 Preferably, the solid support is heated in 0.01MNaCl, 0.05M EDTA and 0.1M Tris (pH8.0) in to 95 ℃ 5 minutes. 此加热步骤去除未共价结合的核酸多聚物如PCR产物。 This heating step removes non-covalently bound to a nucleic acid polymer such as PCR products. 在排列双链核酸的情况中,此步骤也具有将双链变成单链形式(变性)的效应。 In the case of the arrangement of the double stranded nucleic acid, this step also has a double-stranded into single-stranded form (denaturation) effect.

排列可通过生物素化的探针(例如寡核苷酸、核酸片段、PCR产物等)加以检测。 Arrangement by biotinylated probes (such as oligonucleotides, nucleic acid fragments, the PCR products, etc.) to be detected. 使核酸生物素化的方法在本领域众所周知并且由Pierce进行了充分描述(抗生物素蛋白-生物素化学:手册,Pierce化学公司,1992,Rockford Illinois)。 Biotinylated nucleic acids are well known and a method fully described in the art by Pierce (Avidin - Biotin Chemistry: Handbook, Pierce Chemical Company, 1992, Rockford Illinois). 探针一般以0.1ng/mL到10μg/ML在包括GuSCN、GuHCl、甲醛等的标准杂交液中使用(参见Van Ness和Chen,核酸研究19:5143-5151,1991)。 Probes generally 0.1ng / mL to 10μg / ML using standard hybridization solution comprises GuSCN, GuHCl, formaldehyde, etc. (see Van Ness and Chen, Nucleic Acids Research 19: 5143-5151,1991).

为检测杂交事件(即生物素的存在),将固体支持物与链霉抗生物素蛋白/辣根过氧化物酶偶联物一起温育。 To detect hybridization events (i.e., the presence of biotin), the solid support with streptavidin-biotin / horseradish peroxidase conjugate is incubated. 这样的酶偶联物在商业上可获自例如Vector Laboratories(Burlingham,CA)。 Such enzyme conjugates are commercially available from, for example, Vector Laboratories (Burlingham, CA). 链霉抗生物素蛋白以高亲和性结合生物素分子从而使辣根过氧化物酶与杂交探针靠近。 Streptavidin-biotin binding with high affinity to the biotin molecule and thereby horseradish peroxidase hybridization probe close. 未结合的链霉抗生物素蛋白/辣根过氧化物酶偶联物用简单洗涤步骤洗去。 Unbound avidin streptavidin / horseradish peroxidase conjugate is washed away in a simple washing step. 然后在过氧化物和适当缓冲液存在的情况下利用沉淀底物检测过氧化物酶的存在。 It is then detected using a precipitating substrate the presence of peroxidase in the presence of peroxide and the appropriate buffers.

对于比色分析基质,点样于反光性表面如硅片上的蓝色酶产物具有与预期相比低许多倍水平的检测(LLD)。 For colorimetric substrate spotted reflective surface such as blue enzyme product on a silicon wafer having a many times lower compared to the expected level of detection (LLD). 此外,LLD对于不同的有色酶产物有很大不同。 In addition, LLD for different colored enzyme products are very different. 如实施例5中所示,每50μM直径点4-甲氧基-napthol(产生沉淀的蓝色产物)的LLD是约1000个分子,而红色沉淀的物质每50μM直径点给出约1000倍更高1,000,000个分子的LLD。 In the embodiment shown, the diameter of each dot 50μM 4-methoxy -napthol 5 (for generating blue product precipitated) is the LLD about 1000 molecules, whereas a red dot diameter 50μM each substance precipitated is given about 1000-fold more LLD high 1,000,000 molecules. 通过用装有可见光源和CCD相机(Princeton Instruments,Princeton,NJ)的显微镜(如可从Zeiss商业上得到的Axiotech显微镜)检测表面来测定LLD。 By equipped with a visible light source and a CCD camera (Princeton Instruments, Princeton, NJ) microscope (e.g., available commercially from Axiotech Zeiss microscope) to determine the detection surface LLD. 一次可扫描约10,000μM×10,000μM的图象。 One can scan the image of about 10,000μM × 10,000μM.

为了用蓝色比色测定方案,表面在酶反应后必须很干净并且硅片或载片必须在干燥状态下扫描。 In order to use the blue colorimetric assay protocol, the surface must be clean after the enzymatic reaction and the wafer or slide must be scanned in a dry state. 此外,酸反应必须在对照点饱和之前终止。 In addition, the acid reaction must be terminated before the control point of saturation. 对于辣根过氧化物酶,这大约是2-5分钟。 For horseradish peroxidase this is approximately 2-5 minutes.

也可以使用碱性磷酸酶或辣根过氧化物酶(HRP)的化学发光底物或HPP或碱性磷酸酶的荧光底物。 You may be used alkaline phosphatase or horseradish peroxidase (HRP) chemiluminescent substrate or fluorogenic substrate HPP or alkaline phosphatase. 实例包括可获自Perkin Elmer的碱性磷酸酶diox底物或可获自JBL Scientific(Scan Luis Obispo,CA)的AttophosHRP底物。 Examples include alkaline phosphatase available from Perkin Elmer diox substrate or obtainable from JBL Scientific (Scan Luis Obispo, CA) in AttophosHRP substrate. 生物分子溶液的自动转移本发明提供一种将生物分子点样到固体支持物上的方法,此方法包括以下步骤:将弹性探头端部浸入生物分子溶液中;将所述端部从所述溶液中移出以使生物分子溶液附着在所述端部上;并且使所述生物分子溶液与固体支持物接触从而将生物分子溶液从所述端部转移到所述固体支持物上。 Automatic transfer of the biomolecule solution of the present invention provides a biomolecules spotted onto the solid support method, the method comprising the steps of: immersing the resilient end of the probe biomolecule solution; the solution from the said end portion removed so that the biomolecule solution onto the said end portion; and the biomolecule solution is contacted with the solid support so that the biomolecule solution is transferred from the end to the solid support. 在优选实施方案中,接触步骤自动进行,换句话说,是可在x,y和z轴上加以控制的精密自动系统。 , The contacting step is automatically performed in the preferred embodiment, in other words, can be a sophisticated automatic control system of the x, y and z axes. 精密Cartesian自动系统将由与适当马达、放大器、移动控制器、个人计算机和软件连接以驱动工作台的精密线性定位工作台组成。 Automatic precision Cartesian system connected by suitable motor, an amplifier, a mobile controller, personal computer and software to drive a precision linear positioning table worktable. 精密线性定位工作台可获自Parker Hannifin公司(Daedel Division,Harrison City,PA)或THK有限公司(日本东京)。 Precision linear positioning stage are available from Parker Hannifin Company (Daedel Division, Harrison City, PA) or THK Co., Ltd. (Tokyo, Japan). 马达、放大器和移动控制器可获自Parker Hannifin公司(DaedelDivision,Harrison City,PA)或Galil Motion Control有限公司(Mountain View,CA)。 Motors, amplifiers and mobile controller available from Parker Hannifin Company (DaedelDivision, Harrison City, PA) or Galil Motion Control Ltd. (Mountain View, CA). 软件将最可能是自定义的并且可用语言如BorlandC++4.5(Borland International Inc.,Scotts Valley,CA)或VisualBasic 4.0(微软公司,Redmond,WA)编写。 The software will most likely be customizable and available languages ​​as BorlandC ++ 4.5 (Borland International Inc., Scotts Valley, CA), or VisualBasic 4.0 (Microsoft Corporation, Redmond, WA) write. 个人计算机可获自众多生产商如Dell计算机公司(Austin,TX)。 Personal computer available from many manufacturers such as Dell Computer Corporation (Austin, TX).

如上所述的弹性探头制备成装在任何类型的接受器中,本发明中有用的机器人含有适当大小的接受器以容纳弹性探头。 Flexible probe prepared as described above mounted in any type of receptacle, the present invention is useful in a robot receptacle containing an appropriate size to accommodate the elastic probe. 优选的接受器由镍-银或青铜制成,然后在坚硬的镍上镀金。 Preferred receptacle of nickel - is made of silver or bronze, and then nickel plated on hard. 优选的接受器设计是直径0.5mm到2.0mm、更优选地1.68毫米的金属管。 The receptacle is preferably designed to 0.5mm diameter 2.0mm, more preferably 1.68 mm metal tubes. 将0.5mm到1mm厚、更优选地0.64mm厚的方线弯入金属管的一端并封闭。 The thickness of 0.5mm to 1mm, more preferably from rectangular wire bent into a 0.64mm thick end of the metal tube and sealed. 每个套壳制备有凹痕和压环以安全地容纳弹性探头。 Each casing was prepared dents and pressure to securely receive an elastic ring probe. 将探头插入接受器中,因此探头管与接受器末端平齐。 The probe is inserted into the receptacle, the probe tube and thus blunt-ended receptacle.

为了排列液体的目的,将安装头部安装到机器人上。 For purposes of alignment of the liquid, the mounting head mounted on the robot. 该头部具有可在不同印迹应用时拆卸的扣栓。 The head has a detachable blotting in different applications snap latch. 例如,通过取下2个螺丝并用一个设计以容纳从384孔板点样的弹性探头的扣栓代替一个设计以容纳从96孔板点样的弹性探头的扣栓可容易地改变扣栓。 For example, a 96-well plate designed to receive from the probe spotted elastic snap latch may be easily changed by removing the fastening bolt with two screws and is designed to accommodate a snap latch plate 384 spotter from the elastic probe instead. 用精密钻出的、双水平的孔与接受器的绕线和弯曲区域适当匹配借助摩擦力将接受器固定在扣栓上。 Precision drilled holes and double horizontal bending region and the winding receptacle appropriately matched to the receptacle by a frictional force on the fixed fastening bolt. 这种设计使得可以容易地更换损伤或操作不灵的接受器和/或弹性探头。 This design makes it possible to easily replace damaged or ineffective operation of the receiver and / or elasticity of the probe. 一旦插入,接受器/弹性探头组件从扣栓向下伸出25mm,从而允许探针到达装有待排列样品液的微量滴定板底部。 Once inserted, the receptacle / probe assembly extending 25mm from the resilient snap latch downward, thereby allowing the probe reaches the bottom of the package to be arranged in a microtiter plate sample liquid. 阵列本发明的生物分子阵列包括包含表面的固体基质,其中表面至少部分覆盖有一层聚(乙烯亚胺)(PEI)。 Biomolecule arrays of the present invention include an array of solid substrate surface, wherein the surface at least partially covered with a layer of poly (ethyleneimine) (PEI). PEI涂层包括多个由连续第二区环绕并与其邻接的分开的第一区。 PEI coating comprises a plurality of second continuous region surrounds the first region and separate adjacent thereto. 第一区有生物分子和PEI,而第二区有PEI并且基本上没有生物分子。 The first region has a biomolecule and PEI, while the second zone is substantially free of the biomolecule and PEI. 优选地,基质是玻璃板或硅板。 Preferably, the substrate is a glass plate or a silicon plate. 然而,如上所述,基质可以是例如石英、金、尼龙-6,6、尼龙或聚苯乙烯以及它们的复合材料。 However, as described above, the matrix may be for example, quartz, gold, nylon-6,6, nylon or polystyrene, and composites thereof.

PEI涂层优选地含有具100-100,000分子量的PEI。 PEI coating preferably contains PEI having a molecular weight of 100 to 100,000. 双功能偶联剂的反应产物可以置于基质表面和PEI涂层之间,其中反应产物与表面和PEI涂层共价结合并将PEI涂层固定在表面上。 The reaction product of a bifunctional coupling agent may be interposed between the substrate surface and the PEI coating, where the reaction product to the surface and covalently immobilized PEI coating to the surface and the PEI coating binding. 双功能偶联剂包含第一和第二反应性功能基团,其中第一反应性功能集团是例如三(O-C1-C5烷基)硅烷,而第二反应性功能集团是例如环氧化合物、异氰酸酯、异硫氰酸酯和酐基团。 Bifunctional coupling agent comprises a first and a second reactive functional group, wherein the first reactive functional group is such as tris (O-C1-C5 alkyl) silane, and the second reactive functional group is an epoxy compound e.g. , isocyanate, isothiocyanate and anhydride group. 优选的双功能偶联剂是2-(3,4-环氧环己基)乙基三甲氧硅烷;3,4-环氧丁基三甲氧硅烷;3-异氰酸根丙基三乙氧硅烷;3-(三乙氧甲硅烷基)-2-甲基丙基琥珀酸酐和3-(2,3-环氧丙氧基)丙基三甲氧硅烷。 Preferred bifunctional coupling agent is 2- (3,4-epoxycyclohexyl) ethyl trimethoxysilane; 3,4-epoxy-butyl-trimethoxysilane; 3- isocyanato propyl triethoxysilane; 3- (triethoxysilyl) -2-methyl-propyl succinic anhydride and 3- (2,3-epoxypropoxy) propyl trimethoxysilane.

本发明的阵列首先含有含生物分子的区域,其中每个区域具有约1,000平方微米-约100,000平方微米的面积。 The first array of the invention comprising biomolecule-containing regions, where each region has a square micrometers to about 1,000 - an area of ​​about 100,000 square microns. 在优选的实施方案中,第一区具有约5,000平方微米-约25,000平方微米的面积。 In a preferred embodiment, the first region of about 5,000 square microns - an area of ​​about 25,000 square microns.

第一区优选地基本上是圆形,圆圈具有约10μm-200μm的平均直径。 The first region is preferably substantially circular, a circle having an average diameter of about 10μm-200μm. 不论是否是圆形的,第一区的边界优选地互相分开(由第二区分开)至少约25μm,然而不大于约1cm(并且优选地不大于约1,000μm)的平均距离。 Whether circular boundary of the first region are preferably separated from each other (separated by the second region) of at least about of 25 m, but not more than about 1cm (and preferably no greater than about 1,000 m) of the average distance. 在优选的阵列中,相邻第一区的边界由平均约25μm-100μm的间隔分开,其中间隔优选地在整个阵列中一致,并且优选地第一区以如附图中所示的重复几何模式定位。 In a preferred array, the boundaries of neighboring first regions are separated by an average distance of about 25μm-100μm, wherein the spacer is preferably uniform throughout the array, and repeating geometric patterns in the first region is preferably as illustrated in the drawings positioning. 在优选的重复几何模式中,所有相邻的第一区由大约相等的间隔(约25μm-约100μm)分开。 In a preferred repeating geometric pattern, all neighboring first regions are separated by approximately equal intervals (about 25μm- about 100μm).

在优选的阵列中,基质上有10-50个第一区。 In preferred arrays, there are a first region on the substrate 10-50. 在另一实施方案中,基质有50-400个第一区。 In another embodiment, the matrix has a first region 50-400. 在另一优选实施方案中,基质上有400-800个第一区。 In another preferred embodiment, there are a first region on the substrate 400-800.

位于第一区的生物分子优选地是核酸多聚物。 Preferably the biomolecule located in the first region is a nucleic acid polymer. 优选的核酸多聚物是有约15-约50个核苷酸的寡核苷酸。 A preferred nucleic acid polymer is about 15 to about 50 nucleotides in length. 生物分子可以是有约50-约1000个核苷酸的扩增反应产物。 Biomolecule may be amplification reaction products from about 50 to about 1000 nucleotides.

在每个第一区中,生物分子优选地以每2,000μm2第一区105-109个生物分子的平均浓度存在。 In each first region, the biomolecule is preferably present at an average concentration of each of a first region of biomolecules 2,000μm2 105-109. 更优选地,生物分子的平均浓度是每2,000μm2107-109个生物分子。 More preferably, the average concentration of biomolecule per 2,000μm2107-109 biomolecules. 在第二区,生物分子优选地以每2000μm2所述第二区少于103个生物分子的平均浓度存在,更优选地以每2000μm2少于102个生物分子的平均浓度存在。 In the second region, the biomolecule is preferably less than the 2000μm2 per second region 103 is present the average concentration of biomolecule, more preferably present at an average concentration of less than 102 per 2000μm2 biomolecules. 最优选地,第二区不含任何生物分子。 Most preferably, the second regions does not contain any biomolecule.

对于许多生物技术应用,本发明有很大的用途,特别是那些涉及利用聚合酶链式反应(PCR),核酸杂交,通过杂交的核酸测序,病毒、细菌或细胞文库的复制开发大规模诊断筛选方法的方法以及任何其它涉及反复将溶液排列到固体表面上的方法。 For many applications of biotechnology, the present invention has great utility, particularly those involving the use of the polymerase chain reaction (the PCR), nucleic acid hybridization, copying the development of large-scale diagnostic screening by nucleic acid sequencing by hybridization, viruses, bacterial cells or libraries and any other methods involving repeated aligned solution onto a solid surface.

本发明的生物分子阵列可以通过标记的生物分子,例如与可切割的标记共价结合的寡核苷酸来探测。 Biomolecule arrays of the present invention may be labeled biomolecules such as binding with a cleavable covalently labeled oligonucleotide probe. 这些标记的生物分子可用于分析方法如寡核苷酸测序和基因表达分析等。 These biomolecules labeled nucleotide sequencing methods can be used to analyze gene expression analysis, such as oligonucleotides and the like. 标记的生物分子的实例和使用其的分析法描述于美国专利申请Nos.08/786,835;08/786,834和08/787,521(各于1997年1月22日提交),以及具有申请Nos.08/898,180;08/898,564和08/898,501的三个美国部分继续专利申请(各于1997年7月22日提交),以及PCT国际公开Nos.97/27331;97/27325和97/27327中。 Examples of the use and analysis thereof is described in U.S. Patent Application Nos.08 / 786,835 labeled biomolecules; 08 / 786,834 and 08 / 787,521 (filed on each 22 January 1997), and having Application Nos.08 / 898,180 ; 08 / 898,564 and 08 / 898,501 is a continuation in part three US patent applications (each filed July 22, 1997), and PCT international Publication Nos.97 / 27331; 97/27325 and 97/27327. 在此完全引用这6个美国专利申请和3个PCT国际公开的全文作为参考。 The Verbatim six and three US patent application PCT International Publication entirety by reference.

此外,本发明的生物分子阵列可以在单元中含有一个以上寡核苷酸序列。 Further, the biomolecule arrays of the present invention may contain more than one oligonucleotide sequence in the cell. 在成分中含有一个以上寡核苷酸序列的生物分子阵列及其使用描述于1997年7月22日提交的题为“排列单元内的多功能性及其用途”的美国临时专利申请No.60/053,436和与其同时提交的类似题目的美国非临时专利申请No.____中,在此完全引用二者的全文作为参考。 U.S. Provisional Patent component contained in more than one oligonucleotide arrays of biomolecules and their use is described nucleotide sequences, entitled 1997, filed on July 22, "and the use thereof in the versatility of the arrangement means" Application No.60 / 053,436 and US non-provisional patent a similar topic with its concurrently filed application No .____, a complete reference in its entirety herein both as a reference.

本发明的生物分子阵列也可用于进行扩增和其它酶反应,如在我们1997年7月22日提交的题为“对核酸阵列进行的扩增和其它酶反应”的美国临时专利申请No.60/053,428和与其同时提交的类似题目的美国非临时专利申请No.____中所述,在此完全引用二者的全文作为参考。 Biomolecule arrays of the invention can also be used for amplification and other enzymatic reactions, such as us entitled July 22, 1997, filed "amplification of nucleic acid arrays and other enzymatic reactions," U.S. Provisional Patent Application No. 60 / 053,428 and U.S. Non-provisional Patent concurrently with similar topics filed in the No .____, both reference in its entirety herein fully by reference.

本发明的生物分子阵列可以按照1997年7月22日提交的题为“将溶液排列到固体支持物上的装置和方法”的美国临时专利申请No.60/053,435和与其同时提交的类似题目的美国非临时专利申请No.____中所述的方法制备,在此完全引用二者的全文作为参考。 Biomolecule arrays of the present invention may be "the solution to the alignment apparatus and method of solid support" according entitled July 22, 1997, filed in the U.S. Provisional Patent Application No.60 / 053,435 filed concurrently with the subject and the like of preparation of U.S. Non-provisional Patent application No .____ is, both reference in its entirety herein fully by reference.

如1997年7月22日提交的题为“使数据相关的计算机方法和系统”的美国临时专利申请No.60/053,429和与其同时提交的类似题目的美国非临时专利申请No.____(在此充分引用二者的全文作为参考)中所述的使数据相关的计算机系统和方法可与本文公开的方法和装置结合使用。 As 1997 entitled, filed July 22, "the data related to methods and computer systems," the US Provisional Patent Application No.60 / 053,429 and US non-provisional patent similar topics filed simultaneously with No .____ (in this both full reference in its entirety herein by reference) method and apparatus according to correlate data in computer systems and methods disclosed herein may be used in combination.

提供以下实施例作为举例说明而不是限制。 The following examples are provided by way of illustration and not limitation. 实施例实施例1一步法制备有PEI涂层的玻片用0.1N乙酸洗玻片,然后用水冲洗直到从玻片上洗出的水的pH与用于冲洗玻片的水的pH相同。 EXAMPLES Example 1 Method with pH-step slide PEI coated slides washed with 0.1N acetic acid, then rinsed with water until the washing water from the slide and the slide pH water for flushing the same. 然后使玻片干燥。 Slides were then allowed to dry.

向95∶5的乙醇∶水溶液中加入足够量的溶于2-丙醇(Gelest,Inc.,Tullytown,PA,目录号SSP060)中的50% w/w三甲氧基甲硅烷基丙基-聚乙烯亚胺(600MW)以达到2% w/w终浓度。 95:5 the ethanol: water was added dissolved in a sufficient amount of 2-propanol (Gelest, Inc., Tullytown, PA, Catalog No. SSP060) of 50% w / w trimethoxy silyl propyl - Poly ethyleneimine (600MW) to achieve a 2% w / w final concentration. 搅拌此2%溶液5分钟后,将玻片浸入溶液中,轻轻搅拌2分钟,然后取出。 After stirring this 2% solution for 5 minutes, the slides were immersed in the solution, gently stirred for 2 minutes and then removed. 为了洗去过多的甲硅烷基化试剂,将玻片浸入乙醇中。 In order to wash away excess silylating agent, the slides immersed in ethanol. 然后使玻片空气干燥。 Slides were then allowed to air dry. 实施例2一步法制备有PEI涂层的硅片用0.1N乙酸洗硅片(WaferNet,San Jose,CA),然后用水冲洗直到从硅片上洗出的水的pH与用于冲洗硅片的水的pH相同。 Method pH wash with PEI coated silicon wafer (WaferNet, San Jose, CA) with 0.1N acetic acid, then rinsed with water until the washing water from the silicon wafer of Example 2 step for flushing with the embodiment of the wafer the same pH of the water. 然后使硅片干燥。 Silicon wafer was then dried.

向95∶5的乙醇∶水溶液中加入足够量的溶于2-丙醇(Gelest,Inc.,Tullytown,PA,目录号SSP060)中的50% w/w三甲氧基甲硅烷基丙基-聚乙烯亚胺(600MW)以达到2% w/w终浓度。 95:5 the ethanol: water was added dissolved in a sufficient amount of 2-propanol (Gelest, Inc., Tullytown, PA, Catalog No. SSP060) of 50% w / w trimethoxy silyl propyl - Poly ethyleneimine (600MW) to achieve a 2% w / w final concentration. 搅拌此2%溶液5分钟后,将硅片浸入溶液中,轻轻搅拌2分钟,然后取出。 After stirring this 2% solution for 5 minutes, the wafer was immersed in the solution, gently stirred for 2 minutes and then removed. 为了洗去过多的甲硅烷基化试剂,将硅片浸入乙醇中。 In order to wash away excess silylating agent, the wafer is immersed in ethanol. 然后使硅片空气干燥。 Silicon wafer was then air dried. 实施例3一步法制备有PEI涂层的玻片用0.1N乙酸洗玻片,然后用水冲洗直到从玻片上洗出的水的pH与用于冲洗玻片的水的pH相同。 Example 3 step synthesis of PEI coated slides provided with slides washed with 0.1N acetic acid, then rinsed with water until a washing from the same slides on the pH of the water used for rinsing slides water pH. 然后使玻片干燥。 Slides were then allowed to dry.

向95∶5的乙醇∶水溶液中加入足够量的亲电子甲硅烷基化试剂,同时搅拌以达到2% w/w终浓度。 95:5 the ethanol: water was added a sufficient amount of an electrophilic silylating agent, while stirring to achieve a 2% w / w final concentration. 亲电子甲硅烷基化试剂是2-(3,4-环氧环己基)乙基三甲氧硅烷(Gelest,Inc.,Catalog No.SIE4670.0),3,4-环氧丁基三甲氧硅烷(Gelest,Inc.,Catalog No.SIE4665.0)或3-异氰酸根丙基三乙氧硅烷(Gelest,Inc.,Catalog No.SII6454.0)之一。 Electrophilic silylating agent is 2- (3,4-epoxycyclohexyl) ethyl trimethoxysilane (Gelest, Inc., Catalog No.SIE4670.0), 3,4- epoxybutyl trimethoxysilane (Gelest, Inc., Catalog No.SIE4665.0) or 3-isocyanato propyl triethoxysilane (Gelest, Inc., Catalog No.SII6454.0) one. 搅拌此2%溶液5分钟后,将玻片浸入溶液中,轻轻搅拌2分钟,然后取出。 After stirring this 2% solution for 5 minutes, the slides were immersed in the solution, gently stirred for 2 minutes and then removed. 为了洗去过多的甲硅烷基化试剂,将玻片浸入乙醇中。 In order to wash away excess silylating agent, the slides immersed in ethanol.

通过用1-甲基-2-吡咯烷酮(NMP)稀释30%聚乙烯亚胺(Polysciences,Warrington,PA)水溶液来制备70,000分子量聚乙烯亚胺的3%(w/v)溶液。 70,000 molecular weight was prepared 3% polyethyleneimine (w / v) solution diluted with 30% polyethyleneimine (Polysciences, Warrington, PA) with an aqueous solution of 1-methyl-2-pyrrolidone (NMP) by. 将处理的玻片浸入3%溶液中并轻轻搅拌24小时。 The slides were immersed in a 3% treatment solution and gently stirred for 24 hours. 为了去除玻片上过多的PEI,将玻片浸入NMP中(2X),然后浸入同时含有0.09MNaCl、50mM TrispH7.6和25mM DETA的0.1%十二烷基磺酸钠水溶液中(2X),然后浸入水中(2X),并且最后浸入乙醇中(1X)。 In order to remove the excess slide PEI, the slides were immersed in NMP (2X), then immersed while 0.1% sodium dodecyl sulfate aqueous 0.09MNaCl, 50mM TrispH7.6 and 25mM DETA in (2X), then immersion in water (2X), and finally immersed in ethanol (1X). 然后使玻片空气干燥。 Slides were then allowed to air dry. 实施例4一步法制备有PEI涂层的硅片按照实施例3中所述用0.1N乙酸洗硅片(WaferNet,San Jose,CA),然后也按照实施例3中所述用甲硅烷基化试剂和PEI处理。 Example 4 step synthesis of PEI coated with silicon wafer washed according to Example (WaferNet, San Jose, CA) using the 3 in 0.1N acetic acid, and also as in Example 3 silylation with the PEI reagents and processing. 实施例5从商业弹性探头制备点样头XP54P弹性探头购自Osby-Barton〔Everett Charles(Pomona,CA)的分公司〕。 Example 5 was prepared from a commercial point of elastic-like probe head XP54P elastic probe available from Osby-Barton [Everett Charles (Pomona, CA)] branch. 将探头“端部向下”对着超细的金刚磨石(DMT Inc.,Miami Lattes,FL)并以轻轻的压力在磨石上横移约0.5cm距离。 Probe "ends down" against the ultrafine diamond grindstone (DMT Inc., Miami Lattes, FL) and light pressure to the grindstone on the traverse distance of about 0.5cm. 通过显微镜观察到由此从点样头的末端去掉约0.005英寸(0.001到0.01英寸)的金属。 Thereby observed to be removed from the end of the print head of about 0.005 inch (0.001 to 0.01 inches) by a metal microscope. 然后通过在皮带上摩擦端部将点样头末端磨光。 It is then polished by rubbing the spotting head end portion of the belt end. 然后用水洗端部。 Then with water end. 在开始使用前或使用之间,将点样头干燥保存或于-20℃保存在50%甘油中。 Before use or use between the print head or stored in dry storage in 50% glycerol at -20 ℃. 实施例6用改进的弹性探头装配排列装置将实施例5中制备的端部安装到布列头部中,布列头部装在可在x,y和z轴上控制的精密自动系统上。 Example 6 Improved resilient mounting arrangement probe apparatus embodiment ends prepared in Example 5 is mounted to the column head cloth, the cloth may be mounted on the column head in a sophisticated automatic control system of the x, y and z axes. 精密Cartesian自动系统将由与适当马达、放大器、移动控制器、个人计算机和软件连接以驱动工作台的线性定位工作台组成。 Automatic precision Cartesian system connected by suitable motor, an amplifier, a mobile controller, personal computer and software to drive the linear positioning table worktable. 精密线性定位工作台可获自Parker Hannifin公司(Daedel Division,Harrison City,PA)或THK有限公司(日本东京)。 Precision linear positioning stage are available from Parker Hannifin Company (Daedel Division, Harrison City, PA) or THK Co., Ltd. (Tokyo, Japan). 马达、放大器和移动控制器可获自Parker Hannifin公司(Daedel Division,Harrison City,PA)或Galil Motion Control有限公司(Mountain View,CA)。 Motors, amplifiers and mobile controller available from Parker Hannifin Company (Daedel Division, Harrison City, PA) or Galil Motion Control Ltd. (Mountain View, CA). 实施例7用亲水性表面促进液体汲取、液体转移和微滴点样按照实施例5将弹性探头端部浸于100mM 1,4-二硫苏糖醇和0.1M的硼酸钠溶液中60分钟。 Example 7 A hydrophilic surface facilitate liquid draw, and transfers the liquid droplet spotting according to Example 5 of the elastic end of the probe is immersed in a sodium borate solution 100mM 1,4- 0.1M dithiothreitol for 60 minutes. 二硫苏糖醇将通过硫醇-金配位与金表面反应以制备金亲水性表面(表面基本上羟化)。 Dithiothreitol through a thiol - reactive ligand gold gold gold surface to produce a hydrophilic surface (surface substantially hydroxylation). 实施例8反应性寡核苷酸的制备将75μl 5'-己胺-GTCATACTCCTGCTTGCTGATCCACATCTG-3'(0.5μg/μl)溶液与5μl 20mg/ml的氰尿酰氯和20μl 1M的硼酸钠在室温反应30分钟。 Prepared by reaction of the oligonucleotide to Example 8 75μl 5'- hexylamine -GTCATACTCCTGCTTGCTGATCCACATCTG-3 '(0.5μg / μl) solution was mixed with 5μl 20mg / ml cyanuric chloride and 20μl 1M sodium borate reacted at room temperature for 30 minutes . 实施例9寡核苷酸的布列溶液制备由12.5μL pH8.3的1M硼酸钠(新鲜制备或从-20℃储液冻融)、50μl 0.1μg/μL的5'己胺寡核苷酸(5'己胺-GTCATACTCCTGCTTGCTGATCCACATCTG-3')、7.5μL乙腈中的15mg/mL氰尿酰氯组成的布列溶液。 Bridgetown solution of Example 9 was prepared oligonucleotides hexylamine oligonucleotide consists of 1M sodium borate 12.5μL pH8.3 (freshly prepared stock solution or from -20 ℃ freezing and thawing), 50μl 0.1μg / μL 5 ' (5 'hexylamine -GTCATACTCCTGCTTGCTGATCCACATCTG-3'), 7.5μL acetonitrile 15mg / mL solution of cyanuric chloride cloth out thereof. 将此混合物在室温下温育30到60分钟。 This mixture was incubated at room temperature for 30 to 60 minutes. 然后向溶液加入155μL 80%甘油并将所得溶液充分混合。 Then 155μL 80% glycerol and the resultant solution was added to the solution mixed thoroughly. 在某些情况下,向溶液加入15μL 10%的NP-40或10%的Tween 20或10%的Triton X-100(Rohm和Hass,费城)。 In some cases, to the solution was added 15 L of 10% NP-40 or 10% or 10% Tween 20 Triton X-100 (Rohm and Hass, Philadelphia). 当排列基质由尼龙或硝酸纤维素膜组成时,向布列溶液加入25μL 5M的NaCl。 When arranged in a matrix of nylon or nitrocellulose membrane composition, NaCl 25μL 5M solution was added to the cloth out. 实施例10PCR扩增子的布列溶液当排列PCR扩增子时,在完成热循环步骤后将2.5μL pH8.3的1M硼酸钠(新鲜制备或从-20℃储液冻融)、50μl 0.1μg/μL的5'己胺寡核苷酸(5'己胺-GTCATACTCCTGCTTGCTGATCCACATCTG-3')、7.5μL乙腈中的15mg/mL氰尿酰氯加入含有PCR内容物的PCR管中。 Bridgetown solution of Example 10PCR amplicon PCR amplicon arrangement when, after completion of the thermal cycling steps of 1M sodium borate 2.5μL pH8.3 (freshly prepared stock solution or from -20 ℃ freezing and thawing), 50μl 0.1 μg / μL 5 'hexylamine oligonucleotide (5' hexylamine -GTCATACTCCTGCTTGCTGATCCACATCTG-3 '), 7.5μL acetonitrile 15mg / mL cyanuric chloride was added to the PCR tube containing the contents of PCR. 将此混合物在室温下温育30到60分钟。 This mixture was incubated at room temperature for 30 to 60 minutes. 然后向溶液加入155μL 80%甘油并将得到的溶液充分混合。 The solution was then mixed thoroughly 155μL 80% glycerol was added to the solution and the resulting. 在某些情况下,向溶液加入15μL 10%的NP-40或10%的Tween 20或10%的Triton X-100。 In some cases, the solution 15 L 10% NP-40 or 10% or 10% Tween 20 Triton X-100 was added. 当排列基质由尼龙或硝酸纤维素膜组成时,向布列溶液加入25μL 5M的NaCl。 When arranged in a matrix of nylon or nitrocellulose membrane composition, NaCl 25μL 5M solution was added to the cloth out. 实施例11排列寡核苷酸的制备按照实施例6将实施例5的改进的弹性探头安装在自动移液装置中,并且按照实施例7处理弹性探头端部。 Embodiment of the arrangement of oligonucleotides in Example 11 was prepared according to Example 6 the resilient end of the probe of Example 5 improved elastic probe installed in the automatic pipetting apparatus, and the process will be implemented as described in Example 7. 将端部浸入实施例9的布列溶液中5毫米2秒种。 The ends of cloth dipped in the solution of Example 9 in column 5 mm Embodiment 2 seconds. 然后机器人用带有溶液的端部以12×6网格在有聚乙烯亚胺(PEI)涂层的硅片上印迹72个微点,硅片根据实施例2、4中的任一项制备或由将会根据合同制备有PEI涂层的基质的Cell Associates(Houston,Texas)等提供。 Then 12 × 6 72 grid imprinted on the microdots (PEI) coated silicon wafer polyethyleneimine robot end with a solution, a silicon wafer prepared according to any one of embodiments 2,4 or by the matrix will be prepared according Cell Associates PEI coated with a contract (Houston, Texas) and the like. 产生的点直径约100-150微米,相邻点之间中心到中心的间距是200微米。 The spot diameter of approx. 100-150 microns spacing between adjacent dots center to center is 200 microns. 实施例12活性PEI位点的封闭为了封闭排列上未反应的PEI位点,用100mg/mL 100% NMP中的琥珀酸酐处理实施例11的阵列15分钟。 Example 12 PEI blocking active sites for closing the embodiment of the unreacted PEI sites on the arrangement, the array processing of Example 11 for 15 minutes 100mg / mL 100% NMP of succinic anhydride. 实施例13未反应的氰尿酰氯位点的封闭为了封闭排列上未反应的氰尿酰氯位点,用0.1M 0.01M Tris中的甘油处理实施例12的阵列15分钟,然后用Tens(0.1MNaCl,0.1% SDS,0.01MTris,5 mM EDTA)洗4次。 Example 13 closed cyanuric chloride sites embodiment of the unreacted cyanuric chloride to the closure unreacted sites on the arrangement, the array of Example 12 treated with 0.1M 0.01M Tris glycerol for 15 min, then treated with Tens (0.1MNaCl , 0.1% SDS, 0.01MTris, 5 mM EDTA) to wash four times. 实施例14杂交方法使实施例13的阵列中的固定寡核苷酸与其生物素化的互补产物(5'-生物素-TGTGGATCAGCAAGCAGGAGTATG-3')在37℃杂交20分钟,用6X Tens、2 X OHS[0.06M Tris,2mM EDTA、5 X Denhardt's溶液、6X SSC(3M NaCl。0.3M柠檬酸钠,pH7.0)、3.68mM N-月桂酰肌氨酸、0.005%NP-40]洗。 Example 14 Hybridization of a complementary product of Example 13 in an array of fixed oligonucleotide to its biotinylated (5'-biotin--TGTGGATCAGCAAGCAGGAGTATG-3 ') at 37 [deg.] C hybridization embodiment for 20 minutes with 6X Tens, 2 X OHS [0.06M Tris, 2mM EDTA, 5 X Denhardt's solution, 6X SSC (3M NaCl.0.3M sodium citrate, pH7.0), 3.68mM N- lauroylsarcosine, 0.005% NP-40] wash.

杂交后,将硅片浸入0.5μg/ml碱性磷酸酶链霉抗生物素蛋白中15分钟,用2X Tens、4X TWS(0.1M NaCl,0.1%Tween20,0.05M Tris)洗。 After hybridization, the wafer was immersed in 0.5μg / ml streptavidin-alkaline phosphatase avidin for 15 minutes, washed with 2X Tens, 4X TWS (0.1M NaCl, 0.1% Tween20,0.05M Tris). 然后用Vector Blue(Vector Laboratories,Burlingame,Califor-nia)(按照试剂盒方法)使微点显色并用CCD相机和显微镜照相。 Then Vector Blue (Vector Laboratories, Burlingame, Califor-nia) (according to the method of the kit) and the color point of the micro-camera with a CCD camera and microscope. 图5显示产生的图象。 Figure 5 shows the image produced. 实施例15单一阵列单元内的多个寡核苷酸浓度均为0.5μl/μl的2种模板寡核苷酸(寡核苷酸#1=5'己胺-TGTGGATCAGCAAGCAGGAGTATG-3',寡核苷酸#2=5'己胺-ACTACTGATCAGGCGCGCCTTTTTTTTTTTTTTTTTTT-3')分别与20μl 1M硼酸钠中的5μl 20mg/ml氰尿酰氯于室温反应30分钟(总反应体积=100μl)。 A plurality of oligonucleotide concentration in the embodiment 15 are a single unit array 0.5μl / μl of the two kinds of template oligonucleotide (oligonucleotide # 1 = 5 'hexylamine -TGTGGATCAGCAAGCAGGAGTATG-3', oligonucleotide acid # = 2 5 'hexylamine -ACTACTGATCAGGCGCGCCTTTTTTTTTTTTTTTTTTT-3') were reacted with 20μl 1M sodium borate 5μl 20mg / ml cyanuric chloride at room temperature for 30 minutes (total reaction volume = 100μl). 从这两个反应制备由56%甘油和这两种寡核苷酸的稀释组合组成的布列溶液(见表1)。 Preparation of cloth from the column by a solution of 56% glycerol and diluted combinations of the two oligonucleotides consisting of two reactions (see Table 1). 将8个点样头各浸入8种布列溶液中5毫米2秒钟。 The print head 8 of each column is immersed in a solution of 8 kinds of cloth 5 mm for 2 seconds. 然后机器人在聚乙烯亚胺(PEI)涂敷的硅片上用带有溶液的端部印迹2套8个各含有72个微点的12×6网格。 Then 2 sets each containing eight microdots 72 12 × 6 grid blotting robot with an end portion in the polyethyleneimine solution (PEI) coated silicon wafer. 每个网格代表单一的布列溶液。 Each column of the grid representing a single cloth solution. 产生的点直径约100-150微米,相邻点之间中心到中心的间距是200微米。 The spot diameter of approx. 100-150 microns spacing between adjacent dots center to center is 200 microns.

排列后,硅片上未反应的PEI位点用100mg/mL 100%N-甲基吡咯烷酮中的琥珀酸酐封闭15分钟,水洗3X。 After alignment, the unreacted PEI sites on the wafer with 100mg / mL 100% N- methylpyrrolidone succinic anhydride in 15 min, washed with water 3X. 未反应的氰尿酰氯位点用0.01M Tris中的0.1M甘油封闭15分钟,Tens(0.1M NaCl,0.i%SDS,0.01M Tris,5 mM EDTA)洗4X。 The unreacted cyanuric chloride sites were blocked for 15 minutes in 0.01M Tris 0.1M glycerol, Tens (0.1M NaCl, 0.i% SDS, 0.01M Tris, 5 mM EDTA) wash 4X. 然后进行两种杂交。 Then two kinds of hybrids.

在第一种杂交中,将一套8种排列的寡核苷酸组合与互补于寡核苷酸#`1的生物素化寡核苷酸(5'-生物素-TGTGGATCAGCAAGCAGGAGTATG-3')杂交。 In the first hybridization, a set of eight permutations and combinations of oligonucleotides complementary to the biotinylated oligonucleotide # 1 'oligonucleotide (5'-biotin--TGTGGATCAGCAAGCAGGAGTATG-3') hybridizes . 在第二种杂交中,将另一套8种排列的寡核苷酸组合与互补于寡核苷酸#`2的生物素化寡核苷酸(5'-生物素-AAAAAAAAAAAAAAAAAAAAGGCGCGCCTGATCAGTAGT-3')杂交。 In the second hybridization, the oligonucleotides in combination with another set of eight permutations with a complementary biotinylated oligonucleotide # 2 'oligonucleotide (5'-biotin--AAAAAAAAAAAAAAAAAAAAGGCGCGCCTGATCAGTAGT-3') hybridization. 杂交于37℃在Hybriwell SealingCovers(Research Products International Corporation,Mount Prospect,Illinois)下同时进行20分钟,用6X Tens、2 X OHS(0.06M Tris,2mMEDTA)、5 X Denhardt's溶液、6X SSC(3M NaCl,0.3M柠檬酸钠,pH7.0)、3.68mM N-月桂酰肌氨酸、0.005%NP-40洗。 Hybridization at 37 [deg.] C is carried out at Hybriwell SealingCovers (Research Products International Corporation, Mount Prospect, Illinois) at the same time for 20 minutes using 6X Tens, 2 X OHS (0.06M Tris, 2mMEDTA), 5 X Denhardt's solution, 6X SSC (3M NaCl, 0.3M sodium citrate, pH7.0), 3.68mM N- lauroylsarcosine, 0.005% NP-40 wash.

杂交后,将硅片浸入0.5μg/ml辣根过氧化物酶链霉抗生物素蛋白中15分钟,用2X Tens、4X TWS(0.1M NaCl,0.1%Tween 20,0.05M Tris)洗。 After hybridization, the wafer was immersed in 0.5μg / ml streptavidin horseradish peroxidase avidin for 15 minutes with 2X Tens, 4X TWS (0.1M NaCl, 0.1% Tween 20,0.05M Tris) wash. 然后用0.4mg/ml的4-甲氧基-1-napthol(0.02%过氧化氢,12%甲醇,PBS)使微点显色,最后水洗3X。 And then 0.4mg / ml of 4-methoxy -1-napthol (0.02% hydrogen peroxide, 12% methanol, PBS) of the micro color points, and finally washed with water 3X.

与寡核苷酸#1的互补产物杂交的混合寡核苷酸系列对于含有最高浓度寡核苷酸#1的网格表现最强的颜色强度而对于含有最低浓度寡核苷酸#1的网格表现最低的颜色强度。 Hybridizing a complementary oligonucleotide # 1 product was mixed oligonucleotides for the grid containing the highest concentration series of oligonucleotide # 1 exhibited the strongest color intensity for containing the lowest concentration of oligonucleotide # 1 Network performance grid lowest color intensity. 而且,与寡核苷酸#2的互补产物杂交的混合寡核苷酸系列对于含有最高浓度寡核苷酸#2的网格表现最强的颜色强度而对于含有最低浓度寡核苷酸#2的网格表现最低的颜色强度(见图6)。 Furthermore, oligonucleotide hybridization product # 2 is complementary to a mixed series of oligonucleotides containing the highest concentration to oligonucleotide # 2 mesh showed the strongest intensity of color and for containing the lowest concentration of oligonucleotide # 2 mesh lowest performance color intensity (see FIG. 6).

表1< Table 1 <

>实施例16测定单元大小的一致性制备由56%甘油和44%用蓝色食物染料染色的水组成的布列溶液。 > Bridgetown water solution prepared in Example 16 consistency measurement unit size of 56% glycerol and 44% blue food dye. 将点样头浸入布列溶液中5毫米2秒钟。 The print head is immersed in a solution of 5 mm column cloth 2 seconds. 然后机器人用带有甘油的端部以12×6网格在硅片上印迹72个微点。 Then 12 × 6 72 micro grid points imprinted on the wafer by the robot end with a glycerol. 产生的点直径约100-150微米,相邻点之间中心到中心的间距是200微米。 The spot diameter of approx. 100-150 microns spacing between adjacent dots center to center is 200 microns. 图7显示机器人产生的网格的CCD相机相片。 Figure 7 shows a photo of the robot generated by the CCD camera grid. 点直径的标准偏差约为15%。 The standard deviation of the spot diameter is about 15%. 实施例17测定排列方法内的可重复性制备由56%甘油、0.01M Tris pH7.2、5mm EDTA、0.01%肌氨酰和1%V/V Fluoresbrite Plain 0.5μM微球体(2.5%Solids-latex)(Polysciences,Warrington,PA)组成的布列溶液。 Example 17 Determination of the reproducibility of the preparation method of arrangement from 56% glycerol, 0.01M Tris pH7.2,5mm EDTA, 0.01% sarkosyl, and 1% V / V Fluoresbrite Plain 0.5μM microspheres (2.5% Solids-latex ) Bridgetown solution (Polysciences, Warrington, PA) thereof. 将点样针头浸入溶液中5毫米5秒钟并且然后用于在玻片上印迹多个微点。 Spotting 5 mm needle dipped into the solution for 5 seconds and then blotted plurality of micro-dots on the slide. 然后用荧光滤镜在荧光下进行显微摄影。 Followed by fluorescence microscopic photography filter under fluoroscopy. 图8显示每个微点(阵列单元)荧光微球体的高度重复性点样(通过肉眼观察测定)。 Figure 8 shows each micro-dots (array unit) is highly reproducible spotted fluorescent microspheres (measured by visual observation). 实施例18测定每单元核酸多聚物的浓度将与点样的寡核苷酸互补的寡核苷酸(5'-德克萨斯红-CAGATGTGGATCAGCAAGCAGGAGTATGAC)与3M硫氰酸胍(GcSCN)、0.01M TrispH7.5、5mM EDTA和0.1%肌氨酰中的阵列杂交。 Example 18 Determination of the concentration of polymer per unit of a nucleic acid with a nucleotide spotted oligonucleotides complementary oligonucleotide (5'-Texas Red -CAGATGTGGATCAGCAAGCAGGAGTATGAC) with 3M guanidine thiocyanate (GcSCN), 0.01 array hybridization M TrispH7.5,5mM EDTA and 0.1% Sarkosyl in. 体积足以覆盖固体支持物〔对于玻片(1×3英寸)为1mL〕。 Volume sufficient to cover the solid support slides for [(1 × 3 inches) 1mL]. 德克萨斯红寡核苷酸的浓度为5μg/ml,并且反应在室温进行。 Texas Red oligonucleotide concentration was 5μg / ml, and the reaction was carried out at room temperature. 使杂交进行30分钟。 The hybridization for 30 minutes. 然后用Tens洗玻片(5X)。 Tens slides were then washed (5X). 然后用1mL洗脱缓冲液(0.005M Tris pH7.6,0.0005M EDTA,0.01%肌氨酰)覆盖玻片并加热到95℃ 2分钟。 Then treated with 1mL cover slides and heated to 95 ℃ 2 minutes Elution buffer (0.005M Tris pH7.6,0.0005M EDTA, 0.01% Sarkosyl). 从玻片上汲取溶液并将其置于黑色微量滴定板中。 Draw the solution from the slide and placed in a black microtiter plate. 在黑色微量滴定板中测定荧光。 Fluorescence was measured in a black microtiter plate. 从温育管汲取溶液(200μL)并将其置于黑色微量滴定板中(Dynatek Laboratories,Chantilly,VA)。 The draw solution from the incubation tubes (200 L) and placed in a black microtiter plate (Dynatek Laboratories, Chantilly, VA). 然后用Fluoroskan II荧光计(Flow Laboratories,McLean,VA)直接对平板读数,对于荧光素用495nm的激发波长并且在520nm处监测发射光,对于德克萨斯红用591nm的激发波长并且在612nm处监测发射光,并且丽丝胺或TAMRA用570nm的激发波长并且在590nm处监测发射光。 Then Fluoroskan II fluorometer (Flow Laboratories, McLean, VA) directly on the plate reader, with excitation wavelength for Fluorescein in 495nm and the emitted light monitored at 520nm, the excitation wavelength of 591nm with Texas Red at 612nm and monitoring the emitted light, and lissamine or TAMRA emitted light monitored at 590nm with excitation wavelength of 570nm and. 洗脱的寡核苷酸的量通过用测得的荧光量〔3.84个相对荧光单位(rfu)〕除以德克萨斯红寡核苷酸的比活(每μg寡核苷酸6.9rfu)加以测定。 The amount of oligonucleotide eluted by the amount of fluorescence measured with a 3.84 [Relative Fluorescence Units (RFU)] divided by the Texas Red oligonucleotide specific activity (per μg oligonucleotide 6.9rfu) to be measured. 因此测得阵列中的每单元中存在108个寡核苷酸。 Thus the presence of test oligonucleotide arrays 108 in each of cells obtained.

从前述应当理解,虽然在此为了举例说明的目的描述了本发明的具体实施例,但在不偏离本发明精神和范围的情况下可进行各种修饰。 It will be appreciated from the foregoing that, although the purpose of illustration specific embodiments described in the present invention, but without departing from the spirit and scope of the present invention can be variously modified. 因此,除了所附的权利要求外,本发明不受其它限制。 Thus, in addition to the appended claims, the present invention is not otherwise limited.

Claims (60)

  1. 1.一种生物分子阵列,包括:包含表面的固体基质;所述表面至少部分覆盖有一层聚(乙烯亚胺)(PEI);所述涂层包括多个由第二区环绕并与其邻接的分开的第一区;所述第一区有生物分子和PEI;并且所述第二区有PEI并且基本上没有生物分子。 An array of biological molecules, comprising: a solid substrate comprising a surface; the surface at least partially covered with a layer of poly (ethyleneimine) (the PEI); said second coating layer comprises a plurality of regions surrounding and adjacent thereto separate first region; said first region has a biomolecule and PEI; and the second zone is substantially free of the biomolecule and PEI.
  2. 2.权利要求1的阵列,其中所述基质是玻璃板。 2. The array of claim 1, wherein said substrate is a glass sheet.
  3. 3.权利要求1的阵列,其中所述基质是硅片。 3. The array of claim 1, wherein said substrate is silicon.
  4. 4.权利要求1的阵列,其中所述基质包含选自玻璃、石英、硅、金、尼龙-6,6、尼龙和聚苯乙烯的物质。 4. The array of claim 1, wherein said matrix comprises a material selected from glass, quartz, silicon, gold, nylon-6,6, nylon and polystyrene material.
  5. 5.权利要求1的阵列,其中所述PEI具有100-100,000的分子量。 5. The array of claim 1, wherein said PEI has a molecular weight of 100 to 100,000.
  6. 6.权利要求1的阵列,其中双功能偶联剂的反应产物位于所述表面和所述PEI涂层之间,所述反应产物与所述表面和所述PEI涂层共价结合。 6. The array of claim 1, wherein the reaction product of a bifunctional coupling agent is located between the surface and the PEI coating, said reaction product with the surface and the PEI coating is covalently bound.
  7. 7.权利要求6的阵列,其中所述双功能偶联剂包含第一和第二反应性功能基团,所述第一反应性功能集团是三(O-C1-C5烷基)硅烷而所述第二反应性功能集团选自环氧化合物、异氰酸酯、异硫氰酸酯和酐。 7. The array of claim 6, wherein said bifunctional coupling agent comprises a first and a second reactive functional group, said first reactive functional group is three (O-C1-C5 alkyl) silane and the said second reactive functional group selected from epoxy compound, an isocyanate, isothiocyanate and anhydride.
  8. 8.权利要求7的阵列,其中所述双功能偶联剂选自2-(3,4-环氧环己基)乙基三甲氧硅烷;3,4-环氧丁基三甲氧硅烷;3-异氰酸根丙基三乙氧硅烷;3-(三乙氧甲硅烷基)-2-甲基丙基琥珀酸酐和3-(2,3-环氧丙氧基)丙基三甲氧硅烷。 8. The array of claim 7, wherein said bifunctional coupling agent is selected from 2- (3,4-epoxycyclohexyl) ethyl trimethoxysilane; 3,4-epoxy-butyl-trimethoxysilane; 3- isocyanato propyl triethoxysilane; 3- (triethoxysilyl) -2-methyl-propyl succinic anhydride and 3- (2,3-epoxypropoxy) propyl trimethoxysilane.
  9. 9.权利要求1的阵列,其中每个所述第一区具有约1,000μm2-100,000μm2的面积。 9. The array of claim 1, wherein each of said first region has an area of ​​approximately 1,000μm2-100,000μm2.
  10. 10.权利要求1的阵列,其中所述面积是约5,000μm2-约25,000μm2。 10. The array of claim 1, wherein said area is about from about 5,000μm2- 25,000μm2.
  11. 11.权利要求1的阵列,其中每个所述第一区是基本上圆形的。 11. The array of claim 1, wherein each of said first region is substantially circular.
  12. 12.权利要求11的阵列,其中所述第一区有10μm-200μm的平均直径。 12. The array of claim 11, wherein said first zone having an average diameter of 10μm-200μm.
  13. 13.权利要求1的阵列,其中所述第一区互相之间由至少约25μm的平均间隔分开。 13. The array of claim 1, wherein the first region with each other separated by at least the average spacing of about 25μm.
  14. 14.权利要求1的阵列,其中相邻的所述第一区由不大于约1cm的平均间隔分开。 14. The array of claim 1, wherein said first region adjacent spacing are separated by an average of no more than about 1cm.
  15. 15.权利要求1的阵列,其中相郐的所述第一区由不大于1,000μm的平均间隔分开。 15. The array of claim 1, wherein said first phase Kuai region separated by an average distance of not more than 1,000μm.
  16. 16.权利要求1的阵列,其中相邻的所述第一区由约25μm-100μm的平均间隔分开。 16. The array of claim 1, wherein said first region adjacent spacing are separated by an average of about 25μm-100μm.
  17. 17.权利要求1的阵列,其中所述第一区以重复几何模式定位。 17. The array of claim 1, wherein the first region is positioned in a repeating geometric pattern.
  18. 18.权利要求17的阵列,其中所述第一区互相间隔大约相等的距离。 18. The array of claim 17, wherein said first zones are spaced a distance approximately equal to each other.
  19. 19.权利要求18的阵列,其中所述距离是约25μm-约100μm。 19. The array of claim 18, wherein said distance is from about 25μm- about 100μm.
  20. 20.权利要求17的阵列,其中所述模式包括圆形的第一区,所述圆形的第一区有约100-约1,000μm的平均直径。 20. The array of claim 17, wherein said pattern comprises circular first regions, said circular first regions had an average diameter of 100 to about 1,000μm.
  21. 21.权利要求1的阵列,有10-50个第一区。 21. The array of claim 1, a first region 10-50.
  22. 22.权利要求1的阵列,有50-400个第一区。 22. The array as claimed in claim 1, there is a first region 50-400.
  23. 23.权利要求1的阵列,有400-800个第一区。 23. The array as claimed in claim 1, with a first region 400-800.
  24. 24.权利要求1的阵列,其中所述生物分子选自包括核酸多聚物的群体。 24. The array of claim 1, wherein the biomolecule comprises a nucleic acid polymer is selected from the group.
  25. 25.权利要求24的阵列,其中所述生物分子是有约15-约50个核苷酸的寡核苷酸。 25. The array of claim 24, wherein said biomolecule is from about 15 to about 50 nucleotides in length.
  26. 26.权利要求24的阵列,其中所述扩增反应产物是有约50-约1,000个核苷酸的核酸多聚物。 26. The array of claim 24, wherein the amplification reaction product is from about 50 to about 1,000 nucleotides in a nucleic acid polymer.
  27. 27.权利要求1的阵列,其中所述生物分子以每2,000μm2第一区105-109个生物分子的平均浓度存在于第一区中。 27. The array of claim 1, wherein the average concentration of biomolecule in a first area of ​​each 2,000μm2 105-109 biomolecules present in the first zone.
  28. 28.权利要求27的阵列,其中所述平均浓度是每2,000μm2107-109个生物分子。 28. The array of claim 27, wherein the average concentration per 2,000μm2107-109 biomolecules.
  29. 29.权利要求1的阵列,其中所述生物分子以每2000μm2所述第二区少于103个生物分子的平均浓度存在于第二区中。 29. The array of claim 1, wherein each of said biomolecule to said second region 2000μm2 less than the average concentration of biomolecule 103 is present in the second region.
  30. 30.一种制备生物分子阵列的方法。 30. A method of preparing an array of biomolecules. 此方法包括:提供包含表面的固体基质,其中聚(乙烯亚胺)(PEI)层覆盖所述表面的至少一部分,所述涂层包括多个由连续的第二区环绕并与其邻接的分开的第一区;将生物分子上样到所述的第一区而保持所述的第二区基本上不含生物分子。 This method comprises: providing a solid substrate comprising a surface, wherein the poly (ethyleneimine) (the PEI) layer covering at least a portion of said surface, said coating comprising a plurality of second continuous regions surrounded by adjacent thereto and separated a first region; biomolecule loaded onto a second region to the first region is maintained substantially free of the biomolecule.
  31. 31.权利要求30的方法,其中所述基质是玻璃板。 31. The method of claim 30, wherein said substrate is a glass sheet.
  32. 32.权利要求30的方法,其中所述基质是硅片。 32. The method of claim 30, wherein said substrate is silicon.
  33. 33.权利要求30的方法,其中所述基质包含选自玻璃、石英、硅、金、尼龙-6,6、尼龙和聚苯乙烯的物质。 33. The method of claim 30, wherein said matrix comprises a material selected from glass, quartz, silicon, gold, nylon-6,6, nylon and polystyrene material.
  34. 34.权利要求30的方法,其中所述PEI具有100-100,000的分子量。 34. The method of claim 30, wherein said PEI has a molecular weight of 100 to 100,000.
  35. 35.权利要求30的方法,其中双功能偶联剂的反应产物位于所述表面和所述PEI涂层之间,所述反应产物与所述表面和所述PEI涂层共价结合。 35. The method of claim 30, wherein the reaction product of a bifunctional coupling agent is located between the surface and the PEI coating, said reaction product with the surface and the PEI coating is covalently bound.
  36. 36.权利要求35的方法,其中所述双功能偶联剂包含第一和第二反应性功能基团,所述第一反应性功能集团是三(O-C1-C5烷基)硅烷而所述第二反应性功能集团选自环氧化合物、异氰酸酯、异硫氰酸酯和酐。 36. The method of claim 35, wherein said bifunctional coupling agent comprises a first and a second reactive functional group, said first reactive functional group is three (O-C1-C5 alkyl) silane and the said second reactive functional group selected from epoxy compound, an isocyanate, isothiocyanate and anhydride.
  37. 37.权利要求36的方法,其中所述双功能偶联剂选自2-(3,4-环氧环己基)乙基三甲氧硅烷;3,4-环氧丁基三甲氧硅烷;3-异氰酸根丙基三乙氧硅烷;3-(三乙氧甲硅烷基)-2-甲基丙基琥珀酸酐和3-(2,3-环氧丙氧基)丙基三甲氧硅烷。 37. The method of claim 36, wherein said bifunctional coupling agent is selected from 2- (3,4-epoxycyclohexyl) ethyl trimethoxysilane; 3,4-epoxy-butyl-trimethoxysilane; 3- isocyanato propyl triethoxysilane; 3- (triethoxysilyl) -2-methyl-propyl succinic anhydride and 3- (2,3-epoxypropoxy) propyl trimethoxysilane.
  38. 38.权利要求30的方法,其中每个所述第一区具有约1,000μm2-100,000μm2的面积。 38. The method of claim 30, wherein each of said first region has an area of ​​approximately 1,000μm2-100,000μm2.
  39. 39.权利要求30的方法,其中所述面积是约5,000μm2-约25,000μm2。 39. The method of claim 30, wherein said area is about from about 5,000μm2- 25,000μm2.
  40. 40.权利要求30的方法,其中每个所述第一区是基本上圆形的。 40. The method of claim 30, wherein each of said first region is substantially circular.
  41. 41.权利要求40的方法,其中所述第一区有10μm-200μm的平均直径。 41. The method of claim 40, wherein said first zone having an average diameter of 10μm-200μm.
  42. 42.权利要求30的方法,其中所述第一区互相之间由至少约25μm的平均间隔分开。 42. The method of claim 30, wherein the first region with each other separated by at least the average spacing of about 25μm.
  43. 43.权利要求30的方法,其中相邻的所述第一区由不大于约1cm的平均间隔分开。 43. The method of claim 30, wherein said first region adjacent spacing are separated by an average of no more than about 1cm.
  44. 44.权利要求30的方法,其中相邻的所述第一区由不大于1,000μm的平均间隔分开。 44. The method of claim 30, wherein said first region adjacent spacing are separated by an average of not more than 1,000μm.
  45. 45.权利要求30的方法,其中相邻的所述第一区由约25μm-100μm的平均间隔分开。 45. The method of claim 30, wherein said first region adjacent spacing are separated by an average of about 25μm-100μm.
  46. 46.权利要求30的方法,其中所述第一区以重复几何模式定位。 46. ​​The method of claim 30, wherein the first region is positioned in a repeating geometric pattern.
  47. 47.权利要求46的方法,其中所述第一区互相间隔大约相等的距离。 47. The method of claim 46, wherein said first zones are spaced a distance approximately equal to each other.
  48. 48.权利要求47的方法,其中所述距离是约25μm-约100μm。 48. The method of claim 47, wherein said distance is from about 25μm- about 100μm.
  49. 49.权利要求46的方法,其中所述模式包括圆形的第一区,所述圆形的第一区有约100-约1,000μm的平均直径。 49. The method of claim 46, wherein said pattern comprises circular first regions, said circular first regions had an average diameter of 100 to about 1,000μm.
  50. 50.权利要求30的方法,有10-50个第一区。 50. The method as claimed in claim 30, a first region 10-50.
  51. 51.权利要求30的方法,有50-400个第一区。 51. The method as claimed in claim 30, there is a first region 50-400.
  52. 52.权利要求30的方法,有400-800个第一区。 52. The method as claimed in claim 30, there is a first region 400-800.
  53. 53.权利要求30的方法,其中所述生物分子是核酸多聚物。 53. The method of claim 30, wherein said biomolecule is a nucleic acid polymer.
  54. 54.权利要求53的方法,其中所述生物分子是有约15-约50个核苷酸的寡核苷酸。 54. The method of claim 53, wherein said biomolecule is from about 15 to about 50 nucleotides in length.
  55. 55.权利要求53的方法,其中所述扩增反应产物是有约50-约1,000个核苷酸的核酸多聚物。 55. The method of claim 53, wherein the amplification reaction product is from about 50 to about 1,000 nucleotides in a nucleic acid polymer.
  56. 56.权利要求30的方法,其中所述生物分子以每2,000μm2第一区105-109个生物分子的平均浓度存在于第一区中。 56. The method of claim 30, wherein the average concentration of biomolecule in a first area of ​​each 2,000μm2 105-109 biomolecules present in the first zone.
  57. 57.权利要求56的方法,其中所述平均浓度是每2,000μm2107-109个生物分子。 57. The method of claim 56, wherein the average concentration per 2,000μm2107-109 biomolecules.
  58. 58.权利要求30的方法,其中所述生物分子以每2000μm2所述第二区少于103个生物分子的平均浓度存在于第二区中。 58. The method of claim 30, wherein each of said biomolecule to said second region 2000μm2 less than the average concentration of biomolecule 103 is present in the second region.
  59. 59.权利要求30的方法,其中所述上样生物分子包括将核酸多聚物转移到弹性探头端部上。 59. The method of claim 30, wherein said biological sample on a nucleic acid molecule comprising the elastic polymer transferred onto the probe tip.
  60. 60.权利要求30的方法,其中所述样生物分子包括将核酸多聚物从喷黑印迹头中喷出。 60. The method of claim 30, wherein the biomolecule comprises a nucleic acid-like polymer discharged from the discharge head dark blots.
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